CN105821078A - MGMT-Based method for obtaining high yield of recombinant protein expression - Google Patents
MGMT-Based method for obtaining high yield of recombinant protein expression Download PDFInfo
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Abstract
The present invention relates to an MGMT-Based method for obtaining high yield of recombinant protein expression, and discloses a novel enhancer of protein production in host cells, and a vector for expressing recombinant proteins in these cells, comprising a nucleotide sequence encoding a) a secretion peptidic signal, b) a 6-methylguanine-DNA-methyltransferase enzyme (MGMT, EC 2.1.1.63), a mutant or a catalytic domain thereof, and c) a recombinant protein. Said MGMT enzyme is preferably the so-called SNAP protein.
Description
The application is application number 201180065989.9, filing date December in 2011 9 days, invention entitled " is used for obtaining
The method based on MGMT of high yield expression of recombinant proteins " the divisional application of Chinese patent application.
Technical field
The present invention relates to genetic engineering and biology field.Particularly, the present invention relates to egg in a kind of host cell
White novel enhanced produced.Additionally, the present invention relates to the carrier containing the DNA sequence encoding described enhancer albumen, and
For expressing the purposes of recombiant protein, such as industrial enzymes or pharmaceutical protein, including eucaryon (such as, mammal, such as people) and disease
Toxalbumin.
Background technology
Protein production systems, wherein produces interest polypeptide or albumen in recombinant organisms or cell, is commercial biological skill
The trunk of art.
System the earliest, based on the host such as bacterial expression in escherichia coli, has added and based on eucaryon host is
System, the mammalian cell particularly cultivated, cultivation and the insect cell of whole insecticide form, and transgene mammal
Such as sheep and goat.
Prokaryotic cell culture systems is easily maintained and operates cheaply.But, prokaryotic cell can not carry out turning over of eukaryotic protein
Modify after translating.Additionally, many albumen can not correctly fold, need special program to make it again fold, which increase and produce into
This.
Eukaryotic cell culture system has been described with multiple application.Such as, mammalian cell can be repaiied after translating
Decorations, and generally produce correct that fold and soluble protein.The major defect of mammalian cell system includes needing special
Move with expensive cultivate facility, the risk of infection, this loss that may result in whole culture and potential harmful suckling
The risk of thing protein contamination final products.Insect cell is used alternatingly for express polypeptide.In insect cell used the most universal
Expression system based on baculovirus vector.Under the control of strong natural polyhedrin promoter, by replacing with heterologous gene
Change the baculovirus polyhedrin gene of coding baculovirus major structural protein to build rhabdovirus expression vector.With weight
Papova infects the insect host cell cultivated, and can reclaim consequent albumen from cell itself, if or used
Suitably during secretion signal, from culture medium, reclaim consequent albumen.
But, both systems, all there is expression of recombinant proteins level and the repeatability of quality, that culture infects is relevant
Problem, and need special cultivation facility.Additionally, be used for some protein production and the bar prepared under gmp conditions of having to
Shape viral stocks, the most stable.
Drosophila cell, particularly Drosophila melanogaster (Drosophila melanogaster) S2 cell, by US 5,550,
043, for the expression of albumen disclosed in US 5,681,713 and US 5,705,359.It is different from the baculovirus system of prior art
System, is just provided that protein of interest during the insect cell that only cracking is infected wherein, and method based on S2 cell provides continuous print
The cell expression system of heterologous protein, and therefore produce higher expression.
Have been proposed for several other for strengthening the device that heterologous protein is expressed in host cell: such as, US 5,
919,682 describe and use pCW carrier in prokaryote under the control of tac promoter, coexpression albumen and molecular chaperones
Excess produces the method for functional nitric acid synthase.Additionally, US4,758,512 relate to having specific sudden change in its DNA sequence
The production of host cell, its ability causing organism surface to reveal degraded foreign product reduces.The host organisms of these sudden changes
Can be used for increasing the yield of the foreign protein of genetic engineering.
Vertebrate cells, particularly mammalian cell, be also widely used for the expression of recombiant protein.In cultivating
The amount of the protein production of the cell of growth, over time, depends on some questions, e.g., such as cell density, cell cycle phase,
The cell biological aggregate velocity of albumen, in order to support ambient condition and the cell longevity in cultivation of cell viability and growth
Life (after the most how long, they die from programmed cell death or apoptosis).Such as by controlling nutrient, cell density, oxygen and two
The content of carbonoxide, lactic acid dehydrogenase, pH, permeability, metabolite etc., have been developed for improving cell vigor in cultivation and
The various methods in life-span, and the method improving the yield of desirable proteins.
Other host cell can be used for producing heterologous recombinant protein, particularly plant cell and yeast cells.
Many pharmaceutical proteins in mammal source have been synthesized in plant.These are from blood products, as every in having
Year, more than the human serum albumin of 500 tons of demands, arrives and needs lesser amount of cytokine and other signaling molecule.Great majority are planted
The albumen in thing source produces in transgene tobacco, and directly extracts from leaf.Generally, these albumen are with relatively low
Level produces, typically smaller than the 0.1% of total soluble protein.The most low-level production especially reflects the group of various factors
Close, most important of which is that protein folding and poor stability.Recently, tobacco chloroplast system has been used for higher water-glass
Intelligent's albuminoid (MA JKC et al., 2004).
Yeast system is for producing a large amount of industry and the main matter of bio-pharmaceuticals albumen for many years.Yeast is clearly
The medium of definition can grow into the highest Cytoplasm metric density.Can in yeast overexpression recombiant protein, thus product
Secrete from cell, and can reclaim in fermentation liquid.The albumen of yeast secretary, at consistent glycosylation site, by a large amount of glycosyls
Change.Therefore, recombiant protein expression in Yeast system is always restricted for post-translational glycosylation pattern does not affect albumen merit
Those albumen of energy.Some yeast expression systems are used for expression of recombinant proteins, including Saccharomycodes (Sacharomyces), division
Yeast (Schizosaccharomyces pombe), Pichia sp. (Pichia pastoris) and multiple-shaped nuohan inferior yeast
(Hansanuela polymorpha).Recently, a kind of ability novel having and producing recombinant glycoprotein in yeast is occurred in that
System, its glycosylation sequences is similar to the secreting type human glucoprotein produced in mammalian cell.By eliminating endogenous enzyme (its
High mannose chain is added to N-glycosylation intermediate) modify the glycosylation approach of Pichia sp..Additionally, participate in humanization oligosaccharide
At least five kinds of organized enzymes of chain synthesis are specifically transfected to Pichia sp..A large amount of humanization glycoprotein is produced in yeast
Ability imparts such advantage, and glycosylation structure is high unity and is prone to purification wherein.Furthermore, it is possible to by ferment
Mother uses stream add (fed-batch) to produce, use the fermentation time more shorter than mammalian cell, eliminate by mammal
Virus and the cross-contamination of other mammalian hosts glycoprotein.
But, by using these systems, in the supernatant cultivating cell, produce heterologous protein, phase with about 1-2mg/L
Compared with commercial production purpose, this is at a fairly low.
Therefore, in the urgent need to providing the system of a heterologous protein expression that can reach significantly higher level.
The present invention has catered to this needs, and provides protein expression, and the method has reached than existing protein production
The level of production that means are high 100 times (that is, in supernatant until the albumen of 200mg/L).
The present inventor shows really, uses coding to come from people's 6-methyl guanine-DNA-transmethylase (hMGMT) egg
The nucleotide carrier of white albumen, the albumen in described hMGMT source directly or indirectly connects protein of interest, by described interest
The production of albumen is averagely enhanced to the yield of 40mg/L to 200mg/L.
Accompanying drawing explanation
Figure 1A discloses the schematic diagram of the mRNA of code book invention MGMT fusion protein sequence, from 5' to 3', containing signal
Peptide, MGMT mRNA sequence, intervening sequence, proteolytic cleavage site, recombiant protein gene (exogenous gene), intervening sequence and
Individual labelling (His6) label.
Figure 1B: the DNA of carrier same section and aminoacid sequence, it includes i) insecticide ssBiP signal peptide (italic), ii)
SNAP coding enhancer sequence (Lycoperdon polymorphum Vitt), iii) DNA intervening sequence, iv) enterokinase site coded sequence (runic), v) clone position
The DNA (bold Italic) (also seeing SEQ ID NO:5) of some EcoRV/XmaI (underscore) and vi) coding His label labelling.
Fig. 2 A and Fig. 2 A (Continued) disclose fusion protein S NAP (Lycoperdon polymorphum Vitt) and are connected to the Rift Valley fever virus of His label labelling
The aminoacid sequence of (Rift Valley fever virus) N nucleoprotein (RVF.N, runic), two kinds of albumen are by intervening sequence
GGGS separates.
Fig. 2 B: have on the cell conditioned medium of S2 cell of the DNA vector of SEQ ID NO:19 (SNAP-RVF) in transfection, with or
Stimulate 10 days without cadmium, use anti-HisLabelThe immunoblotting assay of antibody.
Fig. 2 C: with anti-His antibody, at insoluble (INS) or solvable (SOL) protein part of escherichia coli B21 lysate
(fraction) immunoblotting assay carried out on, described antibacterial carries pET302/RVF.N+proTEV+GST plasmid.
Fig. 2 D: immunoblotting assay shows, the secretion chimeric protein SNAP-RVF.N of the S2 cell stimulated from 10 days exist
The amount of the SNAP-RVF.N in continuous part (fraction) sample obtained after two-step purifying, use Talon and
Superdex 75 post.
Fig. 3 A and Fig. 3 A (Continued) disclose fusion protein S NAP (italic) and from west nile virus envelope protein E can
Molten form (Lycoperdon polymorphum Vitt), is connected to DNA and the aminoacid sequence of His label labelling (runic), and albumen is separated by intervening sequence GGGS
(SEQ ID NO:20).
Fig. 3 B: use the immunoblotting assay that anti-His tag antibody is carried out, display to have the present invention to encode SNAP-in transfection
In the supernatant of the S2 cell of the DNA vector (SEQ ID NO:20) of WNsE, stimulate 10 days with or without cadmium, Nile virus envelope
The secretion of the soluble form of albumen E protein.
Fig. 4 A disclose containing BiP peptide signal, MGMT sample coded sequence (SNAP sample), two of IFN α sequence both sides
The schematic diagram of the DNA box of pro-TEV cleavage site (huIFNAI) and His label labelling.
Fig. 4 B and Fig. 4 B (Continued): fusion protein S NAP (Lycoperdon polymorphum Vitt, before be the insecticide peptide signal that represents of italic) and IFN α are (thick
Body) it is followed by DNA and the aminoacid sequence of His label labelling (bold Italic), SNAP and IFN α albumen enterokinase cleavage site point
(underscore) and intervening sequence GGGS separate.
Fig. 4 C: the immunoblotting assay using anti-His tag antibody to carry out, detecting transfection has the present invention to encode IFN α
(S2/SNAP-IFN) expression of IFN α in carrier or the S2 cell conditioned medium of control vector, with or without Cd2+Stimulate.
Fig. 4 D: use anti-SNAP antibody, at the 10 μ LS2/DeSNAPuniv-IFN α cells inducing 10 days with or without cadmium
The immunoblotting assay carried out on supernatant.
Fig. 4 E: infecting the HeLa having the Chikungunya virus (Chikungunya virus) expressing renilla luciferase
The activity of luciferase in cell, with various dose from commercial source (Intergen) or produced by the method for the present invention
Raw IFN α processes described cell.
Fig. 4 F: the work of luciferase in infecting the HeLa cell having the Chikungunya virus expressing renilla luciferase
Property, with the SNAP-IFN α albumen described cell of process obtained by production method of the present invention of various dose.
Fig. 5 represents the different step in recombiant protein production process of the present invention.
Fig. 6 A and Fig. 6 A (Continued) disclose fusion protein S NAP (Lycoperdon polymorphum Vitt, before be insecticide peptide signal) and granzyme M is subsequently
DNA and the aminoacid sequence of His label, SNAP and granzyme M albumen with enterokinase cleavage site point and intervening sequence GGGS every
Open.
Fig. 6 B: the schematic diagram of chimeric fusion protein SNAP-GrM, three potential GrM cleavage sites in prominent SNAP.
Fig. 6 C: the immunoblotting assay using anti-SNAP or anti-His tag antibody to carry out, detecting transfection has the present invention to compile
The expression of SNAP-GrM in the S2 cell conditioned medium of the carrier of code GrM (S2/SNAP-GrM, SEQ ID NO:55).
Fig. 7 A discloses containing BiP sample peptide signal, MGMT coded sequence, cuts at two pro-TEV of IFN α sequence both sides
Cut the schematic diagram of the general DNA box of site (huIFNAI) and His label labelling.
Fig. 7 B and Fig. 7 B (Continued): fusion protein S NAP (Lycoperdon polymorphum Vitt, before be insecticide Bip sample peptide signal) and people's IFN α 1 (amino
Acid is shown in bold) it is followed by DNA and the aminoacid sequence of His label labelling, SNAP and IFN α albumen proTEV cleavage
Point and intervening sequence GGGS separate.
Fig. 7 C: the immunoblotting assay carried out with anti-SNAP antibody, detecting transfection has separately encoded SNAP not have peptide
The carrier (pSNAPf carrier) of signal or separately encoded SNAP, be the carrier (pDV1ssprM-of dengue virus peptide signal before it
SNAP) or coding IFN α include DNA sequence as defined in Fig. 7 A carrier of the present invention (pDeSNAP-4/SNAP-IFNA1,
SEQ ID NO:57) HeLa cell conditioned medium in the expression of SNAP-IFN α.
Fig. 8 A discloses and comprises BiP sample peptide signal, SNAP coded sequence, two pro-TEV cleavage sites, His label marks
Remember, for cloning four unique cloning site BamHI, Eco RV, Xma I and Apa I and the intervening sequence of interest genes
The general DNA box (DeSNAP univ, SEQ ID NO:59 and 60) of GGGS.Need unique site Nhe I of 5' end and 3' end
Not I/Hind III subcloning steps (such as plasmid pcDNA3 or pCI-neo) in mammalian expression vector, and
Need unique site Bgl II of 5' end and the Age I of 3' end for the subcloning steps of spinal animal DES system.
Schematic diagram in Fig. 8 B disclose comprise BiP sample peptide signal, MGMT coded sequence, two pro-TEV cleavage sites,
His label labelling, for clone interest genes four unique cloning site BamHI, Eco RV, Xma I and Apa I and
The general DNA box (DeMGMT univ, SEQ ID NO:69 and 70) of intervening sequence GGGS.
Fig. 9 discloses a kind of mode that exogenous gene inserts DeMGMT Univ.
Figure 10 A discloses the SNAP fusion protein CHIK.sE2-hatched at-80 DEG C, 4 DEG C, 25 DEG C or 37 DEG C 4 days
The heat stability of SNAP, SNAP-WN.EDIII.
Figure 10 B discloses the SNAP fusion protein S NAP-IFN α I hatched at-80 DEG C, 4 DEG C, 25 DEG C or 37 DEG C 4 days
Heat stability.
Figure 10 C: hatch at-80 DEG C, 4 DEG C, 25 DEG C or 37 DEG C bimestrial SNAP fusion protein CHIK.sE2-SNAP,
The heat stability of SNAP-WN.EDIII.
Figure 10 D: hatch the heat of bimestrial SNAP fusion protein S NAP-IFN α I at-80 DEG C, 4 DEG C, 25 DEG C or 37 DEG C
Stability.
Figure 11 A discloses by the carrier of the present invention is introduced S2 cell, with (+) or need not (-) after cadmium induces 10 days,
The production of fusion protein S NAP-SSX2 and SNAP-sFasL in whole supernatants.
Figure 11 B discloses by the carrier of the present invention is introduced S2 cell, with (+) or need not (-) after cadmium induces 10 days,
The production of fusion protein S NAP-sFasL in different piece.
Figure 11 C discloses by the carrier of the present invention is introduced S2 cell, with (+) or need not (-) after cadmium induces 10 days,
The production of fusion protein S NAP-SSX2 in different piece.
Figure 12 A disclose comprise BiP sample peptide signal, MGMT coded sequence (SNAP sample), two of SSX2 cancer antigen both sides
The schematic diagram of the general DNA box of pro-TEV cleavage site and His label labelling.
Figure 12 B: comprise two pro-TEV cleavages of BiP sample peptide signal, MGMT coded sequence, NERMCSL albumen both sides
The schematic diagram of the general DNA box of point and His label labelling.
Figure 12 C: use little mouse-anti SNAP antibody, the immunoblotting carried out on the HeLa cell of transient transfection two days divides
Analysis, display IFN α, the extracellular of SSX2 and NERMCSL or intracellular production.
Figure 13 A discloses and comprises BiP sample peptide signal, MGMT coded sequence (SNAP sample), hSULF-2ΔTMDPolypeptide both sides
The schematic diagram of the general DNA box of two pro-TEV cleavage sites and His label labelling.
Figure 13 B and Figure 13 B (Continued): fusion protein S NAP (Dark grey, before be insecticide BiP sample peptide signal) and hSULF-2ΔTMDIt is followed by DNA and aminoacid sequence, SNAP and hSULF-2 of His label labellingΔTMDAlbumen with proTEV cleavage site and
Intervening sequence GGGS separates.
Figure 13 C: transient transfection has pcDNA3/DeSNAPuniv-hSULF-2ΔTMDSecreted by HEK 293 cell of two days
Secreting type is fitted together to DeSNAP-hSULF-2ΔTMDEnzymatic activity.
Figure 14 A disclose comprise BiP peptide signal, MGMT coded sequence (SNAP sample), two of NERMCSL albumen both sides
The schematic diagram of the DNA box of pro-TEV cleavage site and His label labelling.
Figure 14 B: the immunoblotting assay using anti-SNAP antibody to carry out, detecting transfection has the present invention to encode NERMCSL
The carrier (S2/SNAP-NERMCSL) of albumen or the carrier (CHIK.sE2-of coding Chikungunya virus soluble protein E2
SNAP) in S2 cell conditioned medium, with or without Cd2+Stimulate, the expression of NERMCSL albumen.
Detailed description of the invention
The inventor have observed that 6-methyl guanine-dnmt rna (MGMT) being total to together with interest recombiant protein
Expression drastically increases described recombiant protein at insect cell such as S2 cell and in mammalian cell such as HeLa cell
Production.
6-methyl guanine-DNA-transmethylase (MGMT, also referred to as Atase or AGT, hereinafter referred to as " MGMT ") exists
Numbered EC 2.1.1.63 in IUBMB enzyme nomenclature.It is that the 6-alkylguanine-DNA-alkyl of 207 amino acid residues turns
The DNA moving enzyme repairs enzyme, and it is to repair alkylation DNA in intracellular function.More accurately, MGMT is by by SNIn 2 reactions
Methyl be converted into reactive cysteine residues (cysteine 145) to the O in DNA6-the guanine that methylates plays a role.
Owing to albumen irreversibly inactivates, this repair mechanism is uncommon (Pegg A.E. et al., Mutat.Res.2000；462,
82-100).At present in molecular biology, by with O6The irreversible labelling reaction of-benzyl guanine derivant, this enzyme is used
Come to reporter molecules (Juillerat A. et al., Chemistry&Biology, vol.10,313-on albumen in vivo labelling
317,2003 and WO 2005/085470).
Up to the present, it has been described that be derived from the different enzymes of MGMT (Lim A. et al., EMBO J.15:4050-4060,
1996；Daniels D.S. et al., EMBO J.19:1719-1730,2000；Juillerat A. et al., Chemistry&
Biology,vol.10,313-317,2003、WO 2005/085470、WO 2004/031405).Especially, it is thus achieved that contain
Sudden change Cys62Ala, Lys125Ala, Ala127Thr, Arg128Ala, Gly131Lys, Gly132Thr, Met134Leu,
Arg135Ser, Cys150Ser, Asn157Gly, Ser159Glu, the albumen (WO of 20kDa that is truncated in 182 amino acids
So-called " AGT26 " mutant in 2005/085470, in WO 2006/114409 also referred to as " SNAP 26 ").Specific sudden change
Body " SNAP26 " has been demonstrated the labelling activity with enhancing.But, never show or do not implied, it can strengthen even with it
The expression of the recombiant protein of connection.
Herein the present inventor first propose use MGMT enzyme (EC 2.1.1.63), its mutant, its catalyst structure domain or its
Sub-piece, for strengthening albumen production in host cell, particularly in spinal animal and vertebrate host cell.
When host cell expression includes i) having the peptide secretion signal of function, ii in described host cell) MGMT enzyme, its sudden change
Body, catalyst structure domain or sub-piece and iii) fused polypeptide of protein of interest time, it was observed that potentiation.In order to make enhancing make
With generation, MGMT enzyme (can introduce intervening sequence and other aminoacid) directly or indirectly and be physically connected to protein of interest.It is not subject to
Theoretical constraint, it is contemplated that MGMT enzyme can be as chaperone, such as by being beneficial to from the secretion of host cell and host
The fused polypeptide of stable synthesis in cell conditioned medium, or prevent during host cell synthesis and secretion and be metabolized afterwards
Fall.
Further it has been observed that, MGMT has three-dimensional globular structure, and it includes α spiral (Wibley JEA et al., 2000),
This is mutually compatible with the support effect of MGMT.
In the context of the present invention, " host " cell is any cell that can be used for producing recombiant protein, such as " spinal
Animal " (or invertebrates) cell, vertebrate cells, plant cell, yeast cells or prokaryotic cell.Preferably, they
It is non-vertebrates and vertebrate cells.
Spinal animal (also referred to as invertebrates) includes different doors, it is well known that insecticide, Arachnoidea
(Arachnida), Crustachia (Crustacea), Mollusca (Mollusca), Annelida (Annelida), cirrus
Guiding principle (Cirripedia), Radiata (Radiata), Coelenterata (Coelenterata) and Ciliata
(Infusoria).They are divided into more than 30 door now, from simple organism, such as sponge and flatworm to complicated animal, as
Arthropod and Mollusca.In the context of the present invention, spinal zooblast is preferably insect cell, such as fruit bat or mosquito
Daughter cell, more preferably Drosophila S 2 cells.
Can be used as the cell example being derived from vertebrate organism of host cell system include non-human embryonic stem cell or
Its derivant, such as fowl EBX cell；Convert monkey kidney CVI system (COS-7, ATCC CRL 1651) having SV40 sequence；Human embryo
Kidney system (293)；Baby hamster kidney cell (BHK, ATCC CCL 10)；Chinese hamster ovary cell (CHO)；Mouse sertoli cells
[TM4]；Monkey-kidney cells (CVI, ATCC CCL 70)；African green monkey kidney cell (VERO-76, ATCC CRL-1587)；People's cervix uteri
Cancerous cell (HeLa, ATCC CCL 2)；Madin-Darby canine kidney(cell line) (MDCK, ATCC CCL 34)；Buffalo rats liver (BRL 3A,
ATCC CRL 1442)；Human pneumonocyte (W138, ATCC CCL 75)；Human liver cell (Hep G2, HB 8065)；Mammary gland of mouse swells
Oncocyte (MMT 060562, ATCC CCL51)；Rat hepatoma cell [HTC, Ml.5]；YB2/O(ATCC n℃RL1662)；
NIH3T3；HEK and TRI cell.In the context of the present invention, vertebrate cells be preferably EBX, CHO, YB2/O, COS,
HEK, NIH3T3 cell or derivatives thereof.
In the context of the present invention, available plant cell be tobacco cultivars Bright Yellow 2 (BY2) and
Nicotiana tabacum 1 (Nicotiana Tabaccum) (NT-1).
In the context of the present invention, available yeast cells is: saccharomyces cerevisiae (Saccharomyces
Cerevisiae), fusion yeast (Schizosaccharomyces pombe) and multiple-shaped nuohan inferior yeast (Hansanuela
And (methylotropic yeast) methanotrophic yeast such as Pichia sp. and pichia methanolica polymorpha),
(Pichia methanolica)。
In the context of the present invention, available prokaryote is typically E. coli bacteria or bacillus subtilis
(Bacillus Subtilis) antibacterial.
Therefore, the invention discloses to encode a) preferably there is in spinal zooblast or vertebrate cells merit
Can peptide secretion signal and b) 6-methyl guanine-DNA-transmethylase, its mutant, sub-piece or catalyst structure domain
Polynucleotide expression vector.
Term " carrier " referred to herein as, the DNA of exogenous gene or RNA sequence can introduce whereby in host cell thus turn
Change and promote the medium of the expression of introduced sequence.Carrier can include such as, plasmid, phage and virus, and the most more
Discuss in detail.Can introduce in host cell it is true that any kind of plasmid, cosmid, YAC or viral vector can be used for preparing
Recombinant nucleic acid construct, and in host cell, obtain the expression of protein of interest.Or, wherein it is desirable to certain types of host
When cell expresses protein of interest, it is possible to use selectivity infects required cell type or the viral vector of organization type.At this
In the context of invention, it is also important to note that the carrier for gene therapy (i.e., it is possible to deliver nucleic acid to HOST ORGANISMS and divide
Son).
Such as, viral vector such as slow virus, retrovirus, herpesvirus, adenovirus, adeno-associated virus, poxvirus, bar
Shape virus has the recombinant virus of required cell tropism with other.For building and use the method for viral vector in the art
It is known (seeing Miller and Rosman, BioTechniques, 7:980-990,1992).
Actual preferably viral vector is highly suitable in vertebrates and spinal zooblast in the present invention
Those.
For spinal zooblast, preferred carrier is arbovirus, particularly preferred west nile virus, and it is that segmental appendage moves
Thing carrier.Known is baculovirus by other carrier of effective expression in spinal zooblast.
For vertebrate cells, preferably slow virus, AAV, baculovirus and adenovirus vector.Be suitable in mammal place
The carrier expressed in chief cell can also be non-viral (such as plasmid DNA) source.Suitably plasmid vector includes but not limited to
PREP4, pCEP4 (Invitrogene), pCI (Promega), pCDM8 and pMT2PC, pVAX and pgWiz.
For prokaryote, preferred plasmid, phage and cosmid vector.Suitable carrier for prokaryotic cell system
Include but not limited to pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript (Stratagene), p Poly,
pTrc；pET 11d；pIN；With pGEX carrier.
For plant cell, preferred plasmid expression vector such as Ti-plasmids, and virus expression carrier such as cauliflower mosaic virus
And tobacco mosaic virus (TMV) TMV (CaMV).
Recombiant protein expression in yeast cells can be implemented by the carrier of three types: integration vector (YIp),
Plasmid episomal (YEp) and centriole plasmid (YCp): the suitable carrier for expressing in yeast (such as saccharomyces cerevisiae) includes
But it is not limited to pYepSec1, pMFa, pJRY88, pYES2 (Invitrogen Corporation, San Diego, Calif.)
With pTEF-MF (Dualsystems Biotech production code member: P03303).
The carrier that can be used for gene therapy is known in the art.They are such as slow virus, retrovirus, adenopathy
Poison, poxvirus, herpesvirus, Measles virus, foamy virus or adeno-associated virus (AAV).Viral vector can have duplication energy
Power, or can not there is in heredity replication capacity, thus become replication defective or replicate impaired.Preferably gene therapy carries
Body is the DNA Flap carrier described in WO 1999/055892, US 6,682,507 and WO 2001/27300.
The sequence " encoding " expression product (such as RNA, polypeptide, albumen or enzyme) is, when expressing, causes RNA, polypeptide, egg
The nucleotide sequence that white or enzyme produces；I.e. nucleotide sequence " encodes " the aminoacid sequence of RNA or its coded polypeptide, albumen or enzyme
Row.
In the context of the present invention, " catalyst structure domain " of enzyme refers to the avtive spot of enzyme, or in other words, occurs
The enzyme molecular moiety of substrate catalysis is (herein, at SNIn 2 reactions, methyl group is converted into reactive cysteine residues).Cause
This, term " its catalyst structure domain " is appointed as any fragment or the homologous sequence of MGMT polypeptide, preferably has the sky of at least 80%
So catalysis activity of MGMT enzyme.These fragments (also referred to as " sub-piece ") can include between 20 to 180, preferably 30 to 100
Between aminoacid.The homologous sequence of described catalyst structure domain can have one or more portion causing described catalysis activity
The sudden change divided or all lose.
In the context of the present invention, MGMT enzyme can be that the people MGMT of sequence SEQ ID NO:4 is (with reference to NP_
002403.2), it is accredited as the mice MGMT (SEQ ID NO:45) of NP_032624.1, is accredited as the rat of NP_036993.1
MGMT (SEQ ID NO:46) or its homologous sequence.
Term " homology " refers to the sequence with sequence similarity.Term " sequence similarity ", with its all grammer shapes
Formula, refers to the homogeneity between nucleic acid or aminoacid sequence or degree of correspondence.In the context of the present invention, when at least about
80% or at least about 81% or at least about 82% or at least about 83% or at least about 84% or at least about
85% or at least about 86% or at least about 87% or at least about 88% or at least about 89% or at least about
90% or at least about 91% or at least about 92% or at least about 93% or at least about 94% or at least about
95% or at least about 96% or at least about 97% or at least about 98% or the amino acid similarity of at least about 99%
Time, two aminoacid sequences are " homologies ".Preferably, by using Needleman with Wunsch algorithm to identify similar or homology
Peptide sequence.
Preferably, share at least with SEQ ID NO:4 with the sequence of 6-methyl guanine-dnmt rna homology
The amino acid sequence identity of 64%, the amino acid sequence identity of preferably at least about 65% or the amino of at least about 66%
Acid sequence identity or the amino acid sequence identity of at least about 67% or at least about 68% aminoacid sequence same
Property or the amino acid sequence identity of at least about 69% or the amino acid sequence identity of at least about 70% or at least
The amino acid sequence identity of about 71% or the amino acid sequence identity of at least about 72% or the ammonia of at least about 73%
Base acid sequence identity or the amino acid sequence identity of at least about 74% or at least about 75% aminoacid sequence with
One property or the amino acid sequence identity of at least about 76% or the amino acid sequence identity of at least about 77% or extremely
The amino acid sequence identity of few about 78% or the amino acid sequence identity of at least about 79% or at least 80%
Amino acid sequence identity or the amino acid sequence identity of at least about 81% or the aminoacid sequence of at least about 82%
Homogeneity or the amino acid sequence identity of at least about 83% or the amino acid sequence identity of at least about 84% or
The amino acid sequence identity of at least about 85% or the amino acid sequence identity of at least about 86% or at least about 87%
Amino acid sequence identity or the amino acid sequence identity of at least about 88% or the aminoacid sequence of at least about 89%
Row homogeneity or the amino acid sequence identity of at least about 90% or the amino acid sequence identity of at least about 91% or
The amino acid sequence identity of person at least about 92% or the amino acid sequence identity of at least about 93% or at least about
The amino acid sequence identity of 94% or the amino acid sequence identity of at least about 95% or the amino of at least about 96%
Acid sequence identity or the amino acid sequence identity of at least about 97% or at least about 98% aminoacid sequence same
Property and or the amino acid sequence identity of at least about 99%.In one preferred embodiment, SEQ ID NO:4's is same
Source sequence and SEQ ID NO:4 at least 64%, preferably 70% and more preferably 80% are identical.
Preferred homology MGMT sequence comprises the sudden change described in WO 2005/085470, and its position can be easily
The methionine that exchange correspond to SEQ ID NO:4 the 32nd with the start methionine residue based on SEQ ID NO:4, SNAP26 is residual
(therefore, 31 aminoacid should join in the position disclosed in WO 2005/085470 base, thus obtains in SEQ ID NO:4
Corresponding those).
Preferably, the MGMT homologous sequence that can be used for the present invention correspond to the wild type MGMT sequence of SEQ ID NO:4, its
In between 1 and 30, between preferably 6 and 25, and especially 14,15,16,17,18,19,20,21,22 or 23 amino
Sour by other aminoacid replacement, and/or at 1 to 40, C-end, preferably 1 to 20, especially 10 to 20 aminoacid, more preferably
15 aminoacid are deleted.
In one preferred embodiment, compared with SEQ ID NO:4, MGMT homologous sequence comprises following sudden change:
(A) Lys31 is substituted by Arg or Met32 and is substituted by Ser or Cys93 and is substituted by Ala or Lys156 and is substituted by
Ala or Ala158 be substituted by Thr or Arg159 be substituted by Ala, Gly162 be substituted by Lys or Gly163 be substituted by Thr or
Met165 be substituted by Leu or Arg166 be substituted by Ser or Cys181 be substituted by Ser or Asn188 be substituted by Gly or
Ser190 be substituted by Glu or Gly214 be substituted by Pro or Ser215 be substituted by Ala or Ser216 be substituted by Gly or
Gly217 be substituted by Ile or Leu218 be substituted by Gly or Gly220 be substituted by Pro or Ala221 be substituted by Gly or
Trp222 is substituted by Ser, or
(B) Lys31-Met32 is substituted by Arg-Ser or Ala158-Arg159 and is substituted by Thr-Ala or Gly162-
Gly163 is substituted by Lys-Thr or Met165-Arg166 and is substituted by Leu-Ser or Gly162-Gly163/Met165-
Arg166 is substituted by Lys-Thr/Leu-Ser or Asn188/Ser190 and is substituted by Gly/Glu or Gly214-Ser215-
Ser216-Gly217-Leu218 is substituted by Pro-Ala-Gly-Ile-Gly or Gly220-Ala221-Trp222 and is substituted by
Pro-Gly-Ser, preferably with (A) described in other aminoacid replacement any combined, or
(C) block after Leu223 (aminoacid 224-238 be deleted), preferably with (A) or (B) described in any other
Aminoacid replacement is combined.
Preferably MGMT homologous sequence is those being truncated after Leu223.
Preferably MGMT homologous sequence is 2 those modified occurring wherein modifying in (B), optionally after Leu223
Block.
Preferably MGMT homologous sequence is 3 those modified occurring wherein modifying in (B), optionally after Leu223
Block.
Preferably MGMT homologous sequence is 4 those modified occurring wherein modifying in (B), optionally after Leu223
Block.
Preferably MGMT homologous sequence is 5 those modified occurring wherein modifying in (B), optionally after Leu223
Block.
Preferably MGMT homologous sequence is 6 those modified occurring wherein modifying in (B), optionally after Leu223
Block.
Other preferred MGMT homologous sequence be containing 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,
18, those of the combination of the sudden change of 19 or 20 modifications disclosed in (A), and optionally block after Leu223.
In particular it is preferred that containing sudden change Lys31Arg, Met32Ser, Cys93Ala, Lys156Ala, Ala158Thr,
Arg159Ala、Gly162Lys、Gly163Thr、Met165Leu、Arg166Ser、Cys181Ser、Asn188Gly、
Ser190Glu、Gly214Pro、Ser215Ala、Ser216Gly、Gly217Ile、Leu218Gly、Gly220Pro、
Ala221Gly, Trp222Ser and the homologous sequence (that is, the SNAP sequence of SEQ ID NO:2) blocked after Leu223.
In a preferred embodiment, MGMT enzyme is SNAP mutain or its congener of SEQ ID NO:2.
The SNAP mutant of SEQ ID NO:2 and people's 6-methyl guanine-DNA-transmethylase (NP_002403.2, SEQ ID NO:
4) aminoacid sequence shares the homology of 77%, with mice 6-methyl guanine-DNA-transmethylase (NP_032624.1,
SEQ ID NO:45) aminoacid sequence share 70% homology.
Preferably, the SNAP albumen of the homologous sequence of described SNAP albumen and sequence SEQ ID NO:2 more than at least 80%,
Preferably 81%, more preferably 82%, more preferably 83%, more preferably 84%, more preferably 85%, preferably 86%, more preferably 87%, more excellent
Select 88%, more preferably 89%, more preferably 90%, more preferably 91%, more preferably 92%, more preferably 93%, more preferably 94%, more excellent
Select 95%, more preferably 96% to even more preferably 97% identical.
Preferably, the polynucleotide expression vector of the present invention also includes so that the allogeneic dna sequence of coding protein of interest can
Carry out the cloning site that frame is inserted into.
As represented by the present invention, term " peptide secretion signal " represents short (3-60 the aminoacid instructing albumen to transport
Long) peptide chain.
The example of the secretion signal being applicable to the present invention includes but not limited to the signal peptide sequence (US of mating factor (MF) α
5,879,926)；Invertase (WO 84/01153)；PHO5(DK 3614/83)；YAP3 (yeast aspartic protease 3；WO
95/02059)；With BAR1 (WO 87/02670).
In the context of the present invention, this peptide secretion signal preferably at spinal zooblast or vertebrate cells or
Person has function in the middle of two kinds.
The example of the peptide secretion signal in insect cell with function is: and insecticide ssBiP (SEQ ID NO:48, such as
There is the DNA sequence of SEQ ID NO:11), the BiP sample peptide signal of SEQ ID NO:51 (such as have SEQ ID NO:50's
DNA sequence) and arbovirus present in any peptide signal, such as west nile virus envelope E protein (SEQ ID NO:
15)。
It is interesting that above-mentioned BiP sample peptide signal all has function in spinal animal and vertebrate cells.This
BiP sample signal correspond to the BiP peptide signal of SEQ ID NO:48, and the most last glycine aminoacid be correspond to dengue virus
Aminoacid sequence Pro Thr Ala Leu Ala (SEQ ID NO:61) of E protein cleavage site replaces.Therefore, once albumen
Being translated and be secreted in host cell supernatant, BiP sample signal will advantageously be cut.
Various secretion signals can also be used for the expression in yeast host cell, as in saccharomyces cerevisiae.This includes precursor α
The factor (prepro alpha factor), HSp150, PHO1, SUC2, KILM1 (1 type kills poison toxin) and GGP1.
Cloning site is the convenient sequence being cloned in expression system by the gene of coding protein of interest.It contains restrictive
Site or restricted recognition site, i.e. the position on the DNA molecular containing specific nucleotide sequences, it is by Restriction Enzyme institute
Identify.These are typically palindrome (because Restriction Enzyme combines), and specific Restriction Enzyme usually used as homodimer
Sequence within can cutting its recognition site or between neighbouring two nucleotide.Cloning site is known to those skilled in the art
's.
It is further preferred that polynucleotide expression vector also includes the coding interest heterologous protein being inserted in described cloning site
Or the allogeneic dna sequence of heterologous polypeptide.
Term " allos " refers to the combination of the element of non-naturally-occurring.Such as, the present invention includes encoding " protein of interest/many
Peptide " " allogeneic dna sequence ", these DNA sequence are in the most natively or are positioned at the dye of the host cell for protein expression
In colour solid site.
When the allogeneic dna sequence encoding interest heterologous protein or polypeptide is inserted in the nucleotide carrier of the present invention, excellent
Choosing require its coding include described signal peptide, described MGMT enzyme, its mutant or congener and described interest heterologous protein/
The fused polypeptide of polypeptide.
In a preferred embodiment of the present invention, the DNA sequence encoding described MGMT enzyme is positioned at the described interest of coding
5' or 3' of the DNA sequence of heterologous protein, preferably at 5'.Therefore, MGMT enzyme is connected directly or indirectly to interest heterologous protein/many
Peptide, is preferably placed at the N end of interest heterologous protein/polypeptide.
Particularly preferably, when the active structure domain of interest heterologous protein/polypeptide is positioned at its C-end portion, such as IFN α, compile
The DNA sequence of its described MGMT enzyme of code is positioned at the 5' of the DNA sequence encoding described interest heterologous protein/polypeptide.According to identical
Mode, it can be particularly preferred, and when the active structure domain of interest heterologous protein/polypeptide is positioned at its N-end portion, coding is described
The DNA sequence of MGMT enzyme is positioned at the 3' of the DNA sequence encoding described interest heterologous protein/polypeptide.
More accurately, in the first aspect, the present invention relates to a kind of in host cell, preferably spinal animal
And/or in vertebrate host cell, in insect cell, more preferably express the carrier of recombiant protein, described carrier include according to
The direction of 5' to 3' encodes following nucleotide sequence in single open reading frame:
A) there is in described host cell the peptide secretion signal of function,
B) 6-methyl guanine-DNA-transmethylase (MGMT, EC 2.1.1.63), its mutant or catalyst structure domain,
And
C) recombiant protein.
In the context of the present invention, term " recombiant protein " or " protein of interest " represent that allos produces celliferous base in albumen
Because of product or polypeptide, it is preferably selected from diagnosis and treatment albumen or polypeptide.
It is further preferred that described diagnosis and treatment albumen or polypeptide are selected from as follows:
-antibacterial or the immunogenic protein of virus, the more preferably albumen of (infectious, pathogenic) virus, such as from stepping on
Leather, Japanese encephalitis, Ticks propagate encephalitis, yellow heat, Usu figure, sieve Theo, Mu Lei encephalitis, Wei Saiersi Blang (Wesselbron),
Hereby card or the EDIII albumen of west nile virus, or the nucleoprotein N from Rift Valley fever or toscana virus, or agree from datum hole
The soluble form of the E2 envelope protein of refined virus, or the soluble form of west nile virus E envelope protein, and
-blood factor, anticoagulant, somatomedin, hormone, vaccine, therapeutic enzyme, monoclonal antibody and cytokine
(such as IFN-α, granzyme M and FasL),
-antigen, such as cancer antigen, such as the N-petiolarea of cancer testes antigens SSX2 or ERC/Mesotheline (without corresponding translation)
Territory (NERCMSL),
-anti-tumor protein, such as sulfatase (heparan-sulfate6-O-in FasL, or heparin sulphuric acid 6-O-
Endosulfatases) (hSULF),
-microorganism, virus and/or parasite polypeptide,
-other useful proteins any (such as contactin).
Albumen FasL is a kind of pro apoptotic protein, and it can serve as antitumor agent.It can be such as by SEQ ID NO:88
Coding.
HSulf albumen (or hSULF) is internal regulation heparin sulphuric acid structure and growth and progress to malignant cell have
Sulfatase (Dai et al., 2005) in the heparin of appreciable impact-sulphuric acid 6-O-.In the context of the present invention, preferably hSulf2
Albumen, and more preferably hSulf-2ΔTMD, wherein membrane spaning domain (TMD) is deleted to improve its dissolubility, this mutant
There is aminoacid sequence SEQ ID NO:95, and encoded by such as SEQ ID NO:94.
The carrier of the present invention can also be used for expressing and purification interest peptide and/or polypeptide.In the context of the present invention, term
" peptide " and " polypeptide " is synonym, represents the short polymer (also referred to as " residue ") of the amino acid monomer being connected with peptide bond.These
Polymer preferably comprises the residue within 100, the residue within more preferably 50.
Particularly, the carrier of the present invention can be used for expressing and purification diagnostic antimicrobial polypeptide, such as antibacterial, viral or parasitic
Worm polypeptide.The example of this peptide species be by secreted by antibacterial, virus or parasite or express antigenic peptides, mucin and/or poison
Element.Preferably, described antigenic peptides is by influenza virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis G
Virus, inhibition of HIV, yellow fever virus, dengue virus, Japanese encephalitis virus, Far East Russian encephalitis virus, Usu figure or Xi Niluo are sick
Poison, Rift Valley fever or toscana virus, Chikungunya virus, respiratory syncytial virus, rocio virus, Mu Lei encephalitis,
Wei Saiersi Blang virus, hereby block virus (Zika Virus), lymphocytic choriomeningitis (lymphocytic
Choriomeningitis) virus, human parvovirus, human papillomavirus, human cytomegalic inclusion disease virus or any disease identified
Poison is expressed.Preferably, described antigenic peptides by parasitic protozoa (as Entamoeba histolytica (Entamoeba histolytica), Lan Shi merchant
Flagellate (Giardia lamblia)), anthelmintic (such as nematicide, cestode or trematodiasis) or arthropod be (such as Crustachia, insecticide
Guiding principle, Aranea guiding principle) express.Preferably, described antigenic peptides is by infective bacterial, such as Streptococcus (Streptococcus), staphylococcus
Belong to (Staphylococcus) and escherichia coli expression.Infectious prions is well known in the art.People can enumerate,
As an example, botulic neurotoxin, C. perfringens epsilon toxin, Ricin, saxitoxin, shiga toxin, tetrodotoxin, TTX
Element, staphyloentero-toxin etc..Mucin is also known in the art.MUC5AC, MUC5B and MUC2 are the examples.These are real
It is not restrictive for executing example, and any peptide/polypeptide can be expressed by the method for the present invention.
Contactin is belonging to the molecule subgroup of immunoglobulin superfamily, its nervous system express (see
Shimoda and Watanabe, the summary of 2009).They participate in mental disorder, especially infantile autism.By the system of the present invention
The preferred contactin produced is contactin 2 and contactin 4.Such as contactin 4 (CNTN4) is by SEQ ID NO:91
Coding (correspond to the amino acid/11 9-990 of intact proteins NP_783200.1).
Known a large amount of cancer antigen is the effective vaccine target for treating cancer.Producing (see Cheever in a large number of this peptide species
Et al., the list in 2009) the most very important for obtaining effective cancer vaccine.It is interesting that the load of the present invention
Body is obtained in that the high-caliber restructuring cancer antigen that can be used in immunization therapy or generation antibody or Method for cancer diagnostics.
SSX2 and NERCMSL is two examples of cancer antigen.SSX2 cancer antigen is by the DNA encoding with SEQ ID NO:76
(gene bank: NM_175698).The N-petiolarea (NERCMSL) of ERC/Mesotheline is encoded by SEQ ID NO:83.This anti-
The former detection antigen being commonly used for suffering from the patient of pernicious mesothelium.
The method according to the invention can produce any albumen.
It is preferable, however, that albumen is human cytokines, such as insulin, IFN, FasL, Mesotheline, hSULF or contact egg
In vain.
More generally, optimization protein be up to the present be difficult to mass-produced those.This albumen be such as FasL,
Granzyme M, hSULF, Mesotheline and contactin.
Coding includes described peptide signal, described MGMT enzyme, its mutant or catalyst structure domain and described interest recombiant protein
The DNA sequence of fused polypeptide can may be operably coupled to inducible promoter, wherein inducible promoter is identical host
Cell has as peptide signal function.
It is highly preferred that in the carrier of the present invention, described open reading frame may be operably coupled to inducible promoter, its
Middle inducible promoter has function in identical host cell as peptide signal.
In cell, coded sequence " may be operably coupled to " expression regulation sequence (i.e. transcription and translation regulating and controlling sequence),
When coded sequence is transcribed into RNA by RNA polymerase, RNA then carry out trans RNA montage (if it comprises intron) with
And, if this sequential coding albumen, then translate into this albumen.
" promoter " is that the nucleotide sequence that a kind of thus initiation transcription may be operably coupled to DNA downstream (i.e. exists
The positive-sense strand 3' direction of double-stranded DNA).Visible transcriptional start site in promoter sequence is (such as, by can with s1 nuclease mapping
To be able to conveniently find), and the protein binding domain (consensus sequence) that responsible RNA polymerase combines.
The context of the present invention can be used for control the promoter of gene expression such as at spinal zooblast or vertebra
Zooblast has function.Such as, for spinal zooblast, it is possible to use the regulation sequence of metallothionein gene
(Brinster et al., Nature, 296:39-42,1982).
Preferably, there are the inducible promoter in carrier of the present invention in insect cell, more preferably at drosophila cell
In, there is promoter activity.Such as fruit bat metallothionein promoter (Lastowski-Perry et al.,
J.Biol.Chem.260:1527 (1985))), it instructs high-caliber genetic transcription in the presence of metal is such as copper sulfate.Or,
Can use Drosophila Actin 5C gene promoter, this is a kind of constitutive promoter and need not add metal
(B.J.Bond et al., Mol.Cell.Biol.6:2080 (1986)).The example of other known fruit bat promoter includes such as luring
Conductivity type heat shock (Hsp70) and COPIA LTR promoter.SV40 early promoter provides the table lower than fruit bat metallothionein
Reach level.
Preferably, there are the inducible promoter in carrier of the present invention in Drosophila melanogaster cell, preferably at fruit bat S2
Cell has promoter activity.Such as at Lastowski-Perry et al., J.Biol.Chem.260:1527 fills in (1985)
Divide the metallothionein promoter described.
Be suitable to the promoter of constitutive expression in mammalian cell and include that cytomegalovirus (CMV) starts immediately in early days
Son, adenovirus major late promoter, phosphoglyceric kinase (PGK) promoter, the thymidine of herpes simplex virus (HSV)-1 swash
Enzyme (TK) promoter.The induction type eukaryotic promoter regulated by the compound of exogenous offer includes but not limited to the gold that zinc is induced
Genus sulfoprotein (MT) promoter, dexamethasone (Dex) inducible mouse mammary tumor virus (MMTV) promoter, T7 polymerase open
Subsystem (WO 98/10088), insect moulting hormones promoter, tetracycline repressible type promoter, tetracycline-inducible start
Son, RU486 inducible promoter and rapamycin inducible promoter.
Preferably, there are the promoter in carrier of the present invention in mammalian cell, preferably have in HeLa cell
Promoter activity.Such as SV 40 promoter.
A series of Yeast promoters can be used for expressing protein in yeast host cell.Some such as ADH2, SUC2 are to lure
Conductivity type, and other such as GAPDH is composing type in expression.Other promoter being suitable to express in yeast includes
TEF, PGK, MF α, CYC-1, GAL-1, GAL4, GAL10, PHO5, glyceraldehyde-3-phosphate dehydrogenase (GAP or GAPDH) and ethanol
Dehydrogenase (ADH) promoter.
In order in plant cell, the most frequently used promoter be cauliflower mosaic virus (CaMV) 35S promoter or its
Enhanced edition, but some can be used to substitute promoter, such as hybridization (ocs) 3mas from Semen Maydis and arabidopsis (A.Thaliana)
Promoter or ubiquitin promoter.Being different from these constitutive promoters, rice alpha-amylase RAmy3D promoter is deprived by sugar
Induction (Hellwig S et al., 2004).
The promoter being suitable to express in e. coli host cell includes but are not limited to phage lamba pL and starts
Son, lac, TRP and IPTG induction type pTAC promoter.
Preferably, peptide secretion signal has function with inducible promoter in identical host cell.
It is further preferred that peptide secretion signal and inducible promoter are to have function in Drosophila S 2 cells and vertebrate cells
's.
Term " induction type ", when for promoter, is well known to the skilled person.Substantially, in induction
Expression under the control of type promoter, when the stimulation that response is applied, is turned " on " or increases.The character stimulated is with promoter
Different.In the case of suitably stimulating, some inducible promoter cause seldom or the expression that can't detect (or not
Express).In the case of not stimulating, other inducible promoter causes the constitutive expression that can detect that.Do not stimulating
In the case of, regardless of expression, when existence correctly stimulates, the expression from any inducible promoter is to increase
's.
Once building suitable carrier and be transfected in selected host cell, preferably fruit bat cell line, by adding
The derivant being suitable to inducible promoter carrys out the expression of inducing heterogenous albumen.Such as cadmium or copper are the derivants of Hsp70 promoter.
For constitutive promoter, such as actin 5C promoter, express and need not derivant.
People's MGMT enzyme of the present invention is preferably by people's mgmt gene sequence NM_002412.3, gene I/D 4255 (SEQ ID
NO:3) encode or encoded by optimization SEQ ID NO:68 (including only 50%G/C).But, in the context of the present invention
Its homologous sequence any can be used, if its encoding function MGMT enzyme, its mutant or catalyst structure domain, preferably SEQ ID
NO:4 or SEQ ID NO:2.
The preferred DNA sequence encoding described MGMT mutant is SNAP DNA sequence SEQ ID NO:1, or coding
SEQ ID NO:2 but there is the DNA sequence SEQ ID NO:47 or SEQ ID NO:67 of 51%G/C content.
In yet another embodiment of the present invention, the nucleotide carrier of the present invention encodes a fragment of at least MGMT enzyme
(fragment of such as SEQ ID NO:4), or the fragment (sheet of the MGMT mutant of such as sequence SEQ ID NO:2 of its congener
Section), its factor remaining the level increase at least 0.5 times to obtain than the total length enzyme originated by this fragment increases interest egg
The biological activity of white expression.As an example, the level of production if, with the total length enzyme of SEQ ID NO:4 is 100mg/
L, then any fragment with the SEQ ID NO:4 of at least 50mg/L level of production is (identical with SEQ ID NO:4 total length enzyme
Experiment condition) be included in the present invention within.
In yet another embodiment of the present invention, polynucleotide expression vector encodes at least one peptide cleavage site, and it is excellent
Bit selecting is between MGMT enzyme or its catalyst structure domain and interest recombiant protein.
The cleavage site (also referred to as " peptide cleavage site ") of peptide be by least one protease (such as, serine protease,
Cysteine proteinase etc.) aminoacid sequence that identified.The example of peptide cleavage site is that the enterokinase of SEQ ID NO:62 is cut
Cut site (AspAspAspAspLys/Asp), such as by coded by DNA sequence SEQ ID NO:12.Enterokinase is a kind of silk
Serine protease (EC 3.4.21.9), it is known that nonactive trypsinogen is converted to activity by the C-end of cutting sequence by it
Trypsin: Val--(Asp)4--Lys--Ile--Val～(trypsinogen) → Val--(Asp)4--Lys (hexapeptide)+
Ile--Val～(trypsin).If being four Asp before Lys and not having proline residue below, then enterokinase is relying
Cut after propylhomoserin.
Another kind of useful peptide cleavage site is so-called " TEV protease " cleavage site, and it has aminoacid sequence SEQ
ID NO:53 or SEQ ID NO:65 (Glu Asn Leu Tyr Phe Gln Gly or Ser), it is such as by DNA sequence SEQ
ID NO:52 or SEQ ID NO:66 coding.TEV protease is that marmor erodens (tobacco etch virus) encodes
The common name of the 27kDa catalyst structure domain of nuclear inclusion body albumen.It is commercially available (Invitrogen).
Cleavage site (SEQ ID NO:61) from the film precursor prM of dengue virus serotypes 1 can also be used for the present invention
Carrier in.
In another embodiment, polynucleotide expression vector of the present invention also encodes a labelling, and it is preferably placed at this
The C end (including peptide signal, mgmt protein or its congener and recombiant protein) of recombiant protein in bright fused polypeptide.
In the context of the present invention, " labelling " is specifically designed to and is easy to from the thick lysate of host cell reclaim polypeptide,
And be preferably selected from: fluorescin, polyhistidine (poly-his) or polyhistidyl-glycine (poly-his-gly) label；
Flu HA label；C-myc label HSV gD (gD) label, Flag-peptide, alpha-tubulin epi-position or T7 base
Because of 10 protein peptide tags.However, it is possible to use other labelling any.In a preferred embodiment of the present invention, carrier bag
Include coding and there are six histidine-tagged DNA of SEQ ID NO:14.
In another embodiment, the polynucleotide expression vector of the present invention also encoded interval sequence, it is preferably placed at MGMT
Between enzyme (or its catalyst structure domain) and interest recombiant protein and/or between interest recombiant protein and labelling.
In the context of the present invention, intervening sequence is to include the amino acid whose aminoacid sequence of at least three, is specifically designed to
Space separates two polypeptide (then these polypeptide couple together indirectly) connected.Such intervening sequence can be such as ammonia
Base acid sequence Gly-Gly-Gly-serine (GGGS, SEQ ID NO:63), and encode its DNA interval sequence
Row are SEQ ID NO:13.In the context of the present invention, the most this DNA sequence is referred to as " DNA intervening sequence " and is positioned at volume
Between code MGMT or the DNA of its catalyst structure domain and recombinant DNA sequence, the upstream of the DNA sequence of optimized encoding peptide cleavage site.
Polynucleotide expression vector disclosed in this invention can have sequence SEQ ID NO:9, sequence SEQ ID NO:10 or
SEQ ID NO:64 (correspond to be not inserted into the empty carrier of interest recombination in cloning site).In detailed description of the invention
In, the carrier of the present invention can encode:
-peptide BiP insect signal (being preferably to have function in S2 drosophila cell) as above or BiP sample signal,
The mgmt protein of-SEQ ID NO:4 or the SNAP albumen of SEQ ID NO:2,
-interest recombiant protein,
-enterokinase peptide cleavage site as above or proTEV cleavage site,
-polyhistidine labelling, and
-there are two of aminoacid sequence Gly-Gly-Gly-serine (GGGS, SEQ ID NO:63) between
Every sequence.
In a preferred embodiment, the expression vector codes of the present invention:
The peptide BiP insect signal of-SEQ ID NO:48,
The mgmt protein of-SEQ ID NO:4 or the SNAP albumen of SEQ ID NO:2,
-interest recombiant protein,
The enterokinase peptide cleavage site of-SEQ ID NO:62,
-polyhistidine labelling, and
-there are two intervening sequences of aminoacid sequence Gly-Gly-Gly-serine (GGGS).
Another preferred embodiment in, the expression vector codes of the present invention:
The BiP sample peptide signal of-SEQ ID NO:51,
The mgmt protein of-SEQ ID NO:4 or the SNAP albumen of SEQ ID NO:2,
-interest recombiant protein,
The proTEV peptide cleavage site of-SEQ ID NO:53,
-polyhistidine labelling, and
-there are two intervening sequences of aminoacid sequence Gly-Gly-Gly-serine (GGGS).
Such as, such carrier can include that sequence SEQ ID NO:19 is (when protein of interest is RVF virus nucleoprotein N
Time), SEQ ID NO:20 (when protein of interest is west nile virus nucleoprotein N), SEQ ID NO:21 or 57 or 72 or 74 (when
When protein of interest is IFN α), SEQ ID NO:77,79 or 81 (when protein of interest is cancer antigen SSX2), SEQ ID NO:55
(when protein of interest is granzyme M), SEQ ID NO:89 (when protein of interest is FasL), SEQ ID NO:84 or 86 (when
When protein of interest is cancer antigen NERCMSL) or SEQ ID NO:92 (when protein of interest is contactin CNTN4).
In second aspect, the invention also discloses the carrier for expressing recombiant protein in host cell, it includes
The nucleotide sequence that direction encoding from 5' to 3' is following in single open reading frame:
A) peptide secretion signal,
B) mgmt protein of SEQ ID NO:4 or the SNAP albumen of SEQ ID NO:2,
C) at least one peptide cleavage site,
D) polyhistidine labelling, and
E) at least one intervening sequence.
In one preferred embodiment, described peptide secretion signal is the BiP sample peptide signal of SEQ ID NO:50.
In a preferred embodiment, described carrier comprises the proTEV peptide cleavage of two SEQ ID NO:52
Point and/or two intervening sequences with aminoacid sequence SEQ ID NO:63.
In a concrete preferred implementation, described carrier comprises sequence SEQ ID NO:59 or SEQ ID NO:
69, described sequence is hereinafter referred to as general DeSNAP box " DeSNAP Univ " and DeMGMT box " DeMGMT in this application
Univ”。
These " DeSNAP Univ " (SEQ ID NO:59) and " DeMGMT Univ " (SEQ ID NO:69) is referred to as " logical
With " sequence, because they are inserted in any kind of carrier being specifically designed to transfecting host to produce heterologous protein, i.e.
Vertebrates carrier (such as pcDNA3 or PCI-neo carrier) and spinal animal carrier are (such as the pMT/BiP/ for DES system
V5-HisA, is shown in below embodiment).
Plasmid example including described universal sequence is SEQ ID NO:64 (including the pUC57 of DeSNAP Univ) and SEQ
ID NO:71 (with the pUC57 of DeMGMT Univ).
Once the heterologous sequence of protein of interest is cloned herein, such carrier can advantageously be transfected into vertebrates or
In spinal animal host cell, in order to produce a large amount of protein of interest.
In a third aspect, the present invention is directed to stable transfection and have described DeSNAP Univ or the weight of DeMGMT Univ carrier
Group cell, i.e. transfection have and are included in the expression vector encoding following nucleotide sequence in single open reading frame, from 5' to 3'
Direction:
A) peptide secretion signal,
B) mgmt protein of SEQ ID NO:4 or the SNAP albumen of SEQ ID NO:2,
C) at least one peptide cleavage site,
D) polyhistidine labelling, and
E) at least one intervening sequence,
Each composition is as defined above.
Its plasmid (including the pUC57 of DeSNAP Univ) preferably including SEQ ID NO:64 or SEQ ID NO:71 (band
Have the pUC57 of DeMGMT Univ) or at least nucleotide sequence SEQ ID NO:59 (DeSNAP Univ) or SEQ ID NO:69
(DeMGMT Univ)。
Preferably, in this one side of the present invention, described reconstitution cell is Bacillus coli cells.
Use this reconstitution cell so that amplification and the expression vector of the purification present invention, preferably include SEQ ID NO:59's
The DeMGMT Univ (such as SEQ ID NO:71) of DeSNAP Univ (such as SEQ ID NO:64) or SEQ ID NO:69.
Therefore, (described carrier is such as producing any expression vector of the present invention to present invention is alternatively directed to this reconstitution cell
Upper described) purposes.
The polynucleotide expression vector of the present invention may also comprise codes selection mark and/or the gene of terminator sequence.
The selection marker thing gene that can include construct in is typically those of imparting selectivity phenotype, such as antibiotic resistance
(such as blasticidin S, ampicillin, kanamycin, hygromycin, puromycin, chloromycetin).
In fourth aspect, the present invention relates to fused polypeptide, it is included in host cell, preferably spinal animal or
In vertebrate cells, in insect cell, more preferably there is the peptide secretion signal of function, and 6-methyl bird as above is fast
Purine-DNA-transmethylase (MGMT) (EC 2.1.1.63), its mutant or catalyst structure domain.
In this fused polypeptide, the most described MGMT enzyme is the albumen of SEQ ID NO:4, the SNAP of SEQ ID NO:2
Protein mutant or its congener.
This fused polypeptide the most also includes interest recombiant protein as above, is preferably placed at MGMT enzyme as above
Or the C end of its catalyst structure domain and/or labelling.This labelling is preferably polyhistidine labelling, and is preferably placed at interest restructuring
The C end of albumen.
The fused polypeptide of the present invention can be SEQ ID NO:33 to 43, SEQ ID NO:56 or the ammonia of SEQ ID NO:58
Base acid sequence (when interest recombiant protein is GrM), SEQ ID NO:73 or 75 (when interest recombiant protein is IFN α), SEQ
ID NO:78 or 80 or 82 (when interest recombiant protein is cancer antigen SSX2), SEQ ID NO:85 or 87 are (when interest restructuring egg
When being NERCMSL in vain), SEQ ID NO:90 (when interest recombiant protein is FasL) or SEQ ID NO:93 (when interest recombinate
When albumen is CNTN4).
Do not degrade it is interesting that the fusion protein of the present invention can be stored at 4 DEG C lasting some months.During storage
This stabilization in vitro effect owing to the support effect of mgmt protein and/or can obtain due to the existence of mgmt protein
High concentration (typically at least 40mg/mL).
The more important thing is, stabilize recombiant protein with being combined in the purge process of secretory protein of MGMT.The most once
Being applied in the experimenter of this demand, it may be used for internal stablizing recombiant protein.Will be for strengthening this with MGMT coupling
Plant the means in recombiant protein life-span in vivo.This internal stablizing effect is the most under study for action.
In the 5th aspect, the present invention relates to include the spinal animal recombinant host cell of expression vector of the present invention.
Spinal zooblast can be from insecticide, Arachnoidea, Crustachia, Mollusca, Annelida, cirrus
Any cell of guiding principle, Radiata, Coelenterata and Ciliata.In the context of the present invention, spinal zooblast
Preferably insect cell, such as fruit bat or mosquito cells.They are more preferably Drosophila S 2 cells.
Drosophila S 2 cells is widely described.They are especially suitable for the high yield of albumen and produce, because in room temperature (24 ± 1
DEG C) under they may remain in suspension culture.Culture medium is the heat inactivation tire Sanguis Bovis seu Bubali being supplemented with between 5% and 10% (v/v)
The M of (FBS) clearly3.In a preferred embodiment of the invention, culture medium contains 5%FBS.After induction, cell is cultivated at serum-free
In medium.In such medium, S2 cell can grow in suspension culture, such as in 250mL to 2000mL revolving bottle,
Stir with 50-60rpm.Cell density generally remains in 106With 107Between the every mL of cell.
The invention still further relates to include that the restructuring S2 drosophila cell of expression vector of the present invention, described expression vector include preferably selecting
Nucleotide sequence from following:
-plasmid SEQ ID NO:64 (with the pUC57 of DeSNAP Univ) or the nucleotide sequence being cloned in cell,
Within 9th, it is deposited in the microorganism of country of France with numbering CNCM I-4581 in December in 2011 according to this cell of budapest treaty
Preservation center (CNCM), Institute Pasteur, Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux
No. 25-28,
-include SEQ ID NO:19 carrier or the nucleotide sequence that is cloned in cell, should according to budapest treaty
Cell is deposited in country's Organism Depositary of France (CNCM), Bath on August 19th, 2010 with numbering CNCM I-4357
Moral institute, Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28,
-include SEQ ID NO:22 carrier of the present invention or the nucleotide sequence that is cloned in cell, this cell in
On October 27th, 2010 is deposited in country's Organism Depositary of France (CNCM) with numbering CNCM I-4381, and Pasteur is studied
Institute,
-include SEQ ID NO:21 carrier of the present invention or the nucleotide sequence that is cloned in cell, this cell in
On October 27th, 2010 is deposited in country's Organism Depositary of France (CNCM) with numbering CNCM I-4382, and Pasteur is studied
Institute,
The carrier of-SEQ ID NO:9 or the nucleotide sequence being cloned in cell, this cell is in JIUYUE in 2010 29 days
With numbering CNCM I-4368 be deposited in France country Organism Depositary (CNCM), Institute Pasteur,
-include SEQ ID NO:20 carrier of the present invention or the nucleotide sequence that is cloned in cell, this cell in
Within 2010, JIUYUE is deposited in country's Organism Depositary of France (CNCM) with numbering CNCM I-4369 on 29th, and Pasteur is studied
Institute,
The carrier of-SEQ ID NO:71,
-include SEQ ID NO:57 or 72 or 74 (when protein of interest is IFN α), SEQ ID NO:77,79 or 81 (when
When protein of interest is cancer antigen SSX2), SEQ ID NO:55 (when protein of interest is granzyme M), SEQ ID NO:89 (when
When protein of interest is FasL), SEQ ID NO:84 or 86 (when protein of interest is cancer antigen NERCMSL) or SEQ ID NO:
92 (when protein of interest is contactin CNTN4) or SEQ ID NO:96 are (when protein of interest is hSULF-2ΔTMDTime) this
Bright carrier.
The S2 cell of stable transfection of the present invention is also selected from as follows:
-it is deposited in country's Organism Depositary of France (CNCM) on August 19th, 2010 with numbering CNCM I-4357,
The cell of Institute Pasteur (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
(S2/SNAP+RVF-N),
-it is deposited in country of France Organism Depositary, Pasteur on October 27th, 2010 with numbering CNCM I-4381
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+WN.EDIII*),
-it is deposited in country of France Organism Depositary, Pasteur on October 27th, 2010 with numbering CNCM I-4382
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+IFNAI*),
-within 29th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4368 in JIUYUE in 2010
Cell (the pDe of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
Snap-1),
-within 29th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4369 in JIUYUE in 2010
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
WNsE+SNAP),
-within 5th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4565 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+DEN1.EDIII),
-within 5th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4566 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+DEN2.EDIII),
-within 5th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4567 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+DEN3.EDIII),
-within 5th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4568 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+DEN4.EDIII),
-within 5th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4569 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+YF.EDIII),
-within 5th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4570 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+JE.EDIII),
-within 5th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4571 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+USU.EDIII),
-within 5th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4572 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+TBE.EDIII),
-within 8th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4576 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+MVE.EDIII),
-within 8th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4577 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+Rocio.EDIII),
-within 8th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4578 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+SLE.EDIII),
-within 8th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4579 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+WSL.EDIII),
-within 8th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4580 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+Zika.EDIII),
-within 9th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4581 in December in 2011
The cell of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
(pDeSnapUniv),
-within 9th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4582 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP-MUB70),
-within 9th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4583 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP-SSX2),
-within 9th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4584 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAPuniv-NERCMSL),
-within 9th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4585 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP-huGrM), and
-within 9th, it is deposited in country of France Organism Depositary, Pasteur with numbering CNCM I-4586 in December in 2011
Cell (the S2/ of institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP-proTEV)。
It is plasmid vector (the pMT/BiP/ including SEQ ID NO:19 with the reconstitution cell of numbering CNCM I-4357 preservation
SNAP-RVF.N/His label) stable macrophage drosophila cell system S2, wherein RVF.N be Rift Valley fever virus (RVF) N resist
Former (see Brehin et al., Virology 371:185,2008).
It is to include stablizing of plasmid vector pMT/BiP/V5-His label with the reconstitution cell of numbering CNCM I-4381 preservation
Macrophage drosophila cell system S2, after wherein SEQ ID NO:22 (SNAP/WN.EDIII) is inserted into BiP sequence, wherein
WN.EDIII is the III domain of west nile virus glycoprotein E.
With the reconstitution cell of numbering CNCM I-4382 preservation be include plasmid vector pMT/V5-His label stablize huge biting
Cell drosophila cell system S2, is wherein inserted with SEQ ID NO:21 (BiP/SNAP/IFN α 1).IFN α 1 is SEQ ID NO:32
People α 1 interferon (Mokkim et al., Protein expression purif.63:140,2009).
It is to include containing SEQ ID NO:20 (WN.sE/SNAP/his label) with the reconstitution cell of CNCM I-4369 preservation
The stable macrophage drosophila cell system S2 of plasmid vector pMT/BiP/V5-His label, wherein WN.sE is west nile virus E
The soluble form of envelope protein.
It is to include containing SEQ ID NO:20 (WN.sE/SNAP/his label) with the reconstitution cell of CNCM I-4369 preservation
The stable macrophage drosophila cell system S2 of plasmid vector pMT/BiP/V5-His label, wherein WN.sE is west nile virus E
The soluble form of envelope protein.
It is to include that plasmid vector pMT/BiP/SNAP+DV1.EDIII/His marks with the reconstitution cell of CNCM I-4565 preservation
The stable macrophage drosophila cell system S2 signed, the wherein EDIII albumen of DV1.EDIII encoding dengue virus 1, and there is sequence
SEQ ID NO:27.
It is to include that plasmid vector pMT/BiP/SNAP+DV2.EDIII/His marks with the reconstitution cell of CNCM I-4566 preservation
The stable macrophage drosophila cell system S2 signed, the wherein EDIII albumen of DV2.EDIII encoding dengue virus 2, and there is sequence
SEQ ID NO:28.
It is to include that plasmid vector pMT/BiP/SNAP+DV3.EDIII/His marks with the reconstitution cell of CNCM I-4567 preservation
The stable macrophage drosophila cell system S2 signed, the wherein EDIII albumen of DV3.EDIII encoding dengue virus 3, and there is sequence
SEQ ID NO:29.
It is to include that plasmid vector pMT/BiP/SNAP+DV4.EDIII/His marks with the reconstitution cell of CNCM I-4568 preservation
The stable macrophage drosophila cell system S2 signed, the wherein EDIII albumen of DV4.EDIII encoding dengue virus-4, and there is sequence
SEQ ID NO:30.
It is to include that plasmid vector pMT/BiP/SNAP+YF.EDIII/His marks with the reconstitution cell of CNCM I-4569 preservation
The stable macrophage drosophila cell system S2 signed, wherein the EDIII albumen of YF.EDIII coding yellow fever virus, and there is sequence
SEQ ID NO:31.
It is to include that plasmid vector pMT/BiP/SNAP+JE.EDIII/His marks with the reconstitution cell of CNCM I-4570 preservation
The stable macrophage drosophila cell system S2 signed, wherein the EDIII albumen of JE.EDIII coding Japanese encephalitis virus, and there is sequence
Row SEQ ID NO:25.
It is to include that plasmid vector pMT/BiP/SNAP+USU.EDIII/His marks with the reconstitution cell of CNCM I-4571 preservation
The stable macrophage drosophila cell system S2 signed, wherein the EDIII albumen of USU.EDIII coding Usu figure virus, and there is sequence
Row SEQ ID NO:24.
It is to include that plasmid vector pMT/BiP/SNAP+TBE.EDIII/His marks with the reconstitution cell of CNCM I-4572 preservation
The stable macrophage drosophila cell system S2 signed, wherein the EDIII albumen of TBE.EDIII coding Far East Russian encephalitis virus, and have
There is sequence SEQ ID NO:26.
It is to include that plasmid vector pMT/BiP/SNAP+MVE.EDIII/His marks with the reconstitution cell of CNCM I-4576 preservation
The stable macrophage drosophila cell system S2 signed, wherein the EDIII albumen of MVE.EDIII coding Mu Lei encephalitis.
It is to include plasmid vector pMT/BiP/SNAP+Rocio.EDIII/His with the reconstitution cell of CNCM I-4577 preservation
The stable macrophage drosophila cell system S2 of label, wherein the EDIII albumen of Rocio.EDIII coding rocio virus.
It is to include that plasmid vector pMT/BiP/SNAP+SLE.EDIII/His marks with the reconstitution cell of CNCM I-4578 preservation
The stable macrophage drosophila cell system S2 signed, wherein SLE.EDIII encodes St. Louis encephalitis virus (Saint-Louis
Encephalitis virus) EDIII albumen.
It is to include that plasmid vector pMT/BiP/SNAP+WSL.EDIII/His marks with the reconstitution cell of CNCM I-4579 preservation
The stable macrophage drosophila cell system S2 signed, wherein the EDIII albumen of WSL.EDIII coding Wei Saiersi Blang virus.
It is to include plasmid vector pMT/BiP/SNAP+Zika.EDIII/His with the reconstitution cell of CNCM I-4580 preservation
The stable macrophage drosophila cell system S2 of label, wherein Zika.EDIII coding hereby blocks the EDIII albumen of virus.
Plasmid vector pMT/BiP/SNAP+SSX2/His label is included with the reconstitution cell of CNCM I-4583 preservation
Stablizing macrophage drosophila cell system S2, wherein SSX2 is SEQ ID NO:76.
It is to include plasmid vector pMT/BiP/SNAP+NERCMSL/His label with the reconstitution cell of CNCM I-4584 preservation
Stable macrophage drosophila cell system S2, wherein NERCMSL is SEQ ID NO:83.
It is include plasmid vector pMT/BiP/SNAP+GrM/His label steady with the reconstitution cell of CNCM I-4585 preservation
Determining macrophage drosophila cell system S2, wherein GrM is SEQ ID NO:54.
It is to include stablizing of plasmid vector pMT/BiP/ProTEV/His label with the reconstitution cell of CNCM I-4586 preservation
Macrophage drosophila cell system S2, wherein ProTEV is SEQ ID NO:52.
In the 6th aspect, present invention is alternatively directed to stable transfection has the vertebrates reconstitution cell of expression vector of the present invention.
Preferably, described vertebrates reconstitution cell is mammalian cell, preferably CHO, YB2/O, COS, HEK,
NIH3T3, HeLa cell or derivatives thereof.It is further preferred that in this case, the expression vector of the present invention includes SEQ ID NO:
57 or 72 or 74 (when protein of interest is IFN α), SEQ ID NO:77,79 or 81 (when protein of interest is cancer antigen SSX2),
SEQ ID NO:55 (when protein of interest is granzyme M), SEQ ID NO:89 (when protein of interest is FasL), SEQ ID
NO:84 or 86 (when protein of interest is cancer antigen NERCMSL), SEQ ID NO:92 are (when protein of interest is contactin CNTN4
Time) or SEQ ID NO:96 (when protein of interest is hSULF-2ΔTMDTime).In the 7th aspect, the present invention relates to strengthen restructuring egg
The white method expressed, including by described albumen and peptide secretion signal and enzyme 6-methyl guanine-DNA-transmethylase (MGMT)
(EC 2.1.1.63), its mutant or catalyst structure domain coexpression.Described coexpression preferably enters in spinal zooblast
OK, and more preferably insect cell.
It is further preferred that in the method, MGMT enzyme is albumen or its congener of SEQ ID NO:4, such as SEQ ID NO:2
SNAP albumen or its congener.
In the context of the present invention, " Enhanced expressing " of term heterologous protein refers to that described albumen is at reconstitution cell supernatant
In or the interior expression of cell self, compared to recombinant vector (that is, not coexpression albumen and MGMT or the SNAP albumen of prior art
Carrier) expression of described albumen that obtained and/or secretion, improve at least 2 times, preferably 5 times, more preferably 10 times, and even
More preferably 20 times.In one preferred embodiment, term " Enhanced expressing " refers to likely have carrier of the present invention from transfection
Host cell supernatant in reclaim at least 40mg/L, preferably at least 50mg/L, more preferably at least 60mg/L protein of interest.
Term " coexpression " refers to coding i) recombiant protein, ii) MGMT enzyme, its mutant or catalyst structure domain and iii) peptide
The DNA sequence of secretion signal, may be operably coupled to expression regulation sequence (i.e. transcription and translation regulating and controlling sequence), and is controlled by it
System.Therefore the translation of the DNA sequence of encoded peptide secretion signal, interest heterologous protein and MGMT enzyme causes the formation of fused polypeptide,
Wherein albumen can be separated by intervening sequence as defined above and/or cleavage sites.
" the peptide secretion signal " of fused polypeptide of the present invention be preferably in spinal zooblast or vertebrate cells or
In Liang Zhe and more preferably in insect cell, even more preferably there is in Drosophila S 2 cells the secretion signal of function.
The example of the peptide secretion signal in insect cell with function is: insecticide ssBiP, SEQ of SEQ ID NO:48
The envelope E protein of any peptide signal, such as west nile virus present in the BiP sample signal of ID NO:51 and arbovirus
(SEQ ID NO:15).
The example of the peptide secretion signal in vertebrates and spinal zooblast with function is SEQ ID NO:51
BiP sample signal.
In eighth aspect, the present invention relates to improve the production of interest recombiant protein or in cell is cultivated, produce restructuring egg
White method, including using the carrier of the present invention as above or recombinant host cell as above.
More accurately, improve the production of interest recombiant protein or in cell is cultivated, produce the described method of recombiant protein
Including step:
A) polynucleotide expression vector of the present invention encoding described protein of interest is provided,
B) described expression vector is introduced host cell, preferably spinal animal or vertebrate host cell,
C) nucleotide making the described host cell of introducing carries out expression to produce described interest recombiant protein.
Preferably, described spinal animal host cell is insect cell, such as Drosophila S 2 cells.
Preferably, described vertebrate host cell is mammalian cell, such as CHO, YB2/O, COS, HEK,
NIH3T3, HeLa cell or derivatives thereof.
By making in this way, interest recombiant protein obtains with the cells and supernatant of the recovery of at least 40mg/L or above
To express.
Drosophila cell system S2 in gene outcome direct secretion to medium is the side of being preferable to carry out of the present invention by use
Formula (allows in direct secretion to medium to utilize an efficient step purification system).
In the 9th embodiment, the present invention relates to enzyme 6-methyl guanine-DNA-transmethylase (MGMT) (EC
2.1.1.63), its mutant or catalyst structure domain are used for strengthening recombiant protein preferably has replication form or defective vector in infection
Spinal animal and/or vertebrate host cell, the more preferably level of production in insect cell or mammalian cell
Purposes.
MGMT enzyme can be the people MGMT (with reference to NP_002403.2) of sequence SEQ ID NO:4, be accredited as NP_
The mice MGMT (SEQ ID NO:45) of 032624.1, be accredited as NP_036993.1 rat MGMT (SEQ ID NO:46), its
Homologous sequence or its sub-piece.
Preferably, MGMT mutant enzyme is SNAP albumen or its congener of SEQ ID NO:2, i.e. with sequence SEQ ID
The SNAP albumen of NO:2 is more than at least 80%, preferably 85%, more preferably 90% homogeneity.
Described spinal zooblast is preferably insect cell, such as Drosophila S 2 cells.
In one preferred embodiment, the present invention relates to enzyme 6-methyl guanine-DNA-transmethylase (MGMT)
(EC 2.1.1.63), its mutant or catalyst structure domain are used for strengthening recombiant protein has replication form or defective vector in infection
Vertebrate cells, such as the purposes of the level of production in mammalian cell.
Described vertebrate cells is preferably EBX, CHO, YB2/O, COS, HEK, NIH3T3 cell or derivatives thereof.
Additionally, the present invention relates to encode the DNA sequence of MGMT enzyme, its mutant or catalyst structure domain for improving interest egg
The white purposes of the level of production in reconstitution cell.
The invention still further relates to encode the DNA sequence of MGMT enzyme, its mutant or catalyst structure domain for i) in vitro and in vivo
Stabilized interest recombiant protein, and therefore ii) strengthen the purposes in its in vitro and in vivo life-span.
The most this DNA sequence be people's mgmt gene sequence NM_002412.3, gene I/D 4255 (SEQ ID NO:3) or
Its any homologous sequence of encoding function MGMT enzyme, its mutant or catalyst structure domain (preferably SEQ ID NO:1, SEQ NO:
47, SEQ ID NO:67 or SEQ ID NO:68).
Particularly, the present invention relates to 6-methyl guanine-DNA-transmethylase (MGMT, EC 2.1.1.63), its sudden change
Body or catalyst structure domain, as being fused to or be connected to the protection polypeptide of recombiant protein, improve recombiant protein storage medium,
The purposes of half-life in blood plasma or buffer, improves the purposes of the recombiant protein half-life as medicine or vaccine, or changes
The purposes of the recombiant protein half-life being apt in diagnostic kit.
In the context of the present invention, " improving the level of production " or " the enhancing level of production " of term heterologous protein refers to institute
State albumen in described cell conditioned medium or intracellular expression, (that is, do not include this compared to the recombinant vector of prior art
The carrier of invention) expression of described albumen that obtained, improve at least 2 times, preferably 5 times, more preferably 10 times, even more preferably 20
Times.In one preferred embodiment, term " improvement produces " refers to likely have the host of carrier of the present invention thin from transfection
Born of the same parents' supernatant reclaims at least 40mg/L, preferably at least 50mg/L, more preferably at least 60mg/L protein of interest.
In one preferred embodiment, described recombiant protein is selected from: insulin, IFN, SSX2, granzyme M, FasL,
Mesotheline (NERMCSL), interior sulfatase (hSULF) or contactin.
In a specific embodiment, the invention still further relates to the method for producing interest recombiant protein, method bag
Include step:
A () obtains the allogeneic dna sequence of coding interest recombiant protein；
B described allogeneic dna sequence is inserted the polynucleotide expression vector of the present invention by (), described carrier has such as DNA sequence
Row SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:64 or SEQ ID NO:71,
C polynucleotide transfection host cell (preferably insect cell or mammalian cell) that () obtains by step (b)；
D described polynucleotide that () makes step (c) obtain carry out expression to produce protein of interest；
E () is optional, cut MGMT polypeptide,
F () reclaims protein of interest,
G () is optional, purification protein of interest.
In order to carry out the different step of the inventive method, the conventional molecular biology in the range of art technology can be used
, microbiology and recombinant DNA technology.This technology has complete explanation in the literature.See, such as, Sambrook,
Fitsch&Maniatis, Molecular Cloning:A Laboratory Manual, the second edition (1989) Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. are (herein with reference to " Sambrook et al., 1989 ")；
DNA Cloning:A Practical Approach, Volumes I and II (D.N.Glover edits 1985)；
Oligonucleotide Synthesis (M.J.Gait edits 1984)；Nucleic Acid Hybridization
(B.D.Hames&S.J.Higgins,eds.1984)；Animal Cell Culture (R.I.Freshney edits 1986)；
Immobilized Cells and Enzymes(IRL Press,1986)；B.E.Perbal,A Practical Guide to
Molecular Cloning(1984)；F.M.Ausubel et al., (editor), Current Protocols in Molecular
Biology,John Wiley&Sons,Inc.(1994)。
Term " transfects " and refers to be incorporated into exogenous nucleic acid eukaryotic host cell, thus host cell is introduced by expressing
Gene or sequence, to produce desired substance, are the albumen of introduced gene or sequential coding in the present invention.Accept and express
The host cell of introduced DNA or RNA is " transfected " become " transfectant " or " clone ".It is incorporated in host cell
DNA or RNA can come from any source, including belonging to the cell of same genus or kind with host cell, or belong to and differ genus
Or the cell planted.
In the context of the present invention, can be entered by the classical way in this area with polynucleotide transfection host cell
OK, such as by transfection, infection or electroporation.In another embodiment, can be by liposome transfection (with naked DNA
Form), or with the carrier of other transfection (peptide, polymer etc.) the internal introducing present invention.The cation lipid of synthesis
Can be used for preparing the internal transfection of the gene of coding maker thing liposome (Felgner et al.,
Proc.Natl.Acad.Sci.U.S.A.,84:7413-7417,1987).Can be used for lipid compounds or the combination of transfer nucleic acid
Thing is described in WO 95/18863 and WO 96/17823, and U.S.5, in 459,127.For the purpose of targeting, lipid is chemically
It is coupled to other molecule (see Mackey et al., Proc.Natl.Acad.Sci.U.S.A., 85:8027-8031,1988).Targeting
Peptide such as hormone or neurotransmitter and albumen such as antibody or non-peptide molecule can be with chemical coupling to liposomees.Other molecule also can be used
In promoting nucleic acid transfection in vivo, such as cation oligopeptide (seeing WO95/21931), it is derived from the peptide of DBP (see WO
96/25508) or cationic polymer (see WO 95/21931).It is likely to draw carrier in vivo as naked DNA plasmid
Enter.Means known in the art can be passed through, such as electroporation, microinjection, cell fusion, deae dextran, calcium phosphate precipitation, use
Particle gun or use DNA vector transporter, the naked DNA carrier that will be used for gene therapy introduces in required host cell (see Wu
Et al., J.Biol.Chem., 267:963-967,1992；Wu and Wu, J.Biol.Chem., 263:14621-14624,1988；
Williams et al., Proc.Natl.Acad.Sci.U.S.A., 88:2726-2730,1991).
Term " allow express " polynucleotide are referred to herein as the regulating and controlling sequence being present in carrier and all required compositions
Stimulating (such as activation-inducing type promoter), it is carried out with the amount that be enough to occur polynucleotide to translate.
If necessary, the cutting through in supernatant or restructuring of the MGMT/SNAP polypeptide of produced fusion protein
Intracellular addition has the protease of clear and definite cleavage site and realizes.Such as, by adding enterokinase in reconstitution cell supernatant
And obtain the cutting of enterokinase cleavage site point DDDK/D.Or, MGMT/SNAP polypeptide can be maintained thus strengthen recombiant protein
Life-span.
Additionally, skilled artisan will appreciate that can for maintain or cultivate detection in the culture medium of this host cell express or
The albumen of secretion or polypeptide.Culture medium can be separated with host cell by known method, such as centrifugal or filtration.Then, logical
Cross the known properties feature utilizing albumen or polypeptide, albumen or polypeptide can be detected in cell-free medium.This character can
To include difference immunity, enzyme or the physical property of albumen or polypeptide.Such as, if albumen or polypeptide have the enzymatic activity of uniqueness,
Can be in the analysis to this activity of the host cell used medium enterprising hand-manipulating of needle.Additionally, when obtaining for given albumen
Or during the reactive antibody of polypeptide, (such as, this antibody can be used for detecting albumen or polypeptide in any known immunological method
At Harlowe, et al., 1988, Antibodies:A Laboratory Manual, Cold Spring Harbor
In Laboratory Press).
The recovery of protein of interest is mediated by method as known in the art, includes but not limited to preparative disc gel electricity
Swimming, isoelectrofocusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, precipitation and salting-out chromatdgraphy, extraction and inverse
Stream distribution (countercurrent distribution) etc..Because preferably in connecting markd recombination system of the present invention
Produce protein of interest, described in be marked with help by chromatograph on suitable solid-phase matrix from the thick lysate of host cell reclaim
Polypeptide.Or, the antibody of the polypeptide that the antagonism albumen of generation or antagonism are derived from albumen can be used as recovery catalyst.
Further purification step (g) can be implemented, but what is interesting is and be not required to.
The material being purified can containing less than about 50%, preferably less than about 75%, most preferably less than about 90% with this quilt
The original relevant cell component of material of purification." the purest " represents that use conventional purification technique known in the art can
With the highest purity realized.
In an embodiment of the invention, the method for the present invention allows to obtain in the cells and supernatant reclaimed
Obtain the purest protein of interest of at least 40mg/L, preferably at least 50mg/L, more preferably at least 60mg/L.
(i.e. coupling has the recombiant protein of MGMT/SNAP polypeptide, and it is than list for the interest recombiant protein of the present invention and fusion protein
Only recombiant protein is more stable) can be used in various product.Such as, these restructuring and/or fusion protein can be used for drug regimen
Thing, the treatment such as infected for virus.
In one preferred embodiment, described recombiant protein is selected from: insulin, IFN, FasL, granzyme M, SSX2,
Mesotheline (NERMCSL), interior sulfatase (hSULF) or contactin.
In another embodiment, the present invention provides the infectious viral particle containing above-mentioned nucleic acid carrier.Generally feelings
Under condition, the method such virion of generation by comprising the steps:
A the viral vector of the present invention is incorporated in suitable cell line by (),
B () cultivates described cell line under suitable condition to allow to produce described infectious viral particle,
C () reclaims produced infectious viral particle from the culture of described cell line, and
The infectious viral particle reclaimed described in (d) optionally purification.
When viral vector is deficiency, generally in the complementary cell system of trans offer non-functional viral gene or pass through
Helper virus is used to produce infectious particles.Such as, the appropriate cell line for the adenovirus vector of complementary E1 disappearance includes
293 cells and PER-C6 cell.Infectious viral particle can be reclaimed from culture supernatant or from the cell after cracking.Root
According to standard technique (such as WO96/27677, WO98/00524, WO98/22588, WO98/26048, WO00/40702,
Chromatography described in EP1016700 and WO00/50573, the supercentrifugation in cesium chloride gradient) they can be entered one
Step purification.
Therefore, the present invention relates to include the expression vector of the present invention, recombiant protein, fusion protein, host cell or virus
Granule or the pharmaceutical composition of its combination in any.Such pharmaceutical composition include being mixed with pharmaceutically suitable carrier, by this
The carrier of therapeutic dose, granule, cell or the albumen that bright method obtains.
Parenteral, vein or systemic administration compositions hypodermically can be passed through.When systemic administration, for the present invention
Therapeutic combination be without pyrogen, parenteral acceptable protein solution form.The acceptable albumen of this parenteral is molten
The preparation of liquid, it is contemplated that pH, isotonicity, stability etc., within the skill of the art.
In view of changing pharmaceutically-active many factors, such as state, body weight, the Excreta (sew) of patient and diet, sense
The order of severity, time of application and other clinical factor of dye, dosage will be determined by attending doctor.The medicine of pharmaceutical composition
Carrier and other composition will be selected by those skilled in the art.
Additionally, such as, the fusion of the present invention and recombiant protein can be used as vaccine composition, come seeded with mammalian experimenter with
To viral infection resisting.These albumen can be used alone or be used in combination with other recombiant protein or Therapeutic Vaccination agent.This epidemic disease
The composition of Seedling will be determined by those skilled in the art.
Present invention additionally comprises the fusion protein of the present invention, expression vector, infectious viral particle, host cell or medicine group
Compound is for preparing the purposes in medicine particularly vaccine.
The present invention also provides for the method for treating or prevent human or animal's organism, uses including to described organism
The fusion protein of the present invention of therapeutically effective amount, expression vector, infectious viral particle, host cell or compositions.
The albumen of the last present invention, the especially SNAP albumen of interest fusion protein, can be used as detecting cancer, virus
Infect or the diagnostic agent of antiviral protein antibody existence in biological fluid (such as blood, serum, saliva etc.).These albumen also can be used
The method of virus protein in qualification and/or isolated organism liquid and tissue.Therefore, albumen can be by the examination of this method
Composition in agent box.
Therefore, in one aspect of the method, the invention still further relates to by any method of the present invention obtain from pathogenic or
The recombiant protein of non-pathogenic microorganisms or the recombiant protein of MGMT or SNAP label are used for identifying in biological specimen whether exist
The described pathogenic or purposes of non-pathogenic microorganisms.In one preferred embodiment, described pathogenic microorganism is virus,
The albumen of MGMT or SNAP label is virus protein, as from chikungunya, Dengue, Japanese encephalitis (JE), tick encephalitis
(TBE), yellow heat (YF), Usu figure (USU) or the EDIII of west nile virus or from Rift Valley fever or the core of toscana virus
Albumen N.
In the context of the present invention, described biological specimen refers to blood sample, urine sample or the doubtful any life being infected
Thing sample.
Embodiment
1. plasmid construction
1.1. plasmid pMT/BiP/V5-His A is used.It comprises 3642 nucleotide, and comprises following characteristics:
-metallothionein promoter: base 412-778
-transcription initiation: base 778
-MT forward primer site: base 814-831
-BiP signal sequence: base 851-904 (SEQ ID NO:11)
-multiple clone site: base 906-999
-V5 antigenic tag: base 1003-1044
-polyhistidine district: base 1054-1074
-BGH reverse primer site: base 1094-1111
-SV40 late polyadenylation signal: base 1267-1272
-pUC initial point: base 1765-2438 (complementary strand)
-bla promoter: base 3444-3542 (complementary strand)
-ampicillin (bla) resistant gene ORF: base 2583-3443 (complementary strand)
PUC57Amp carrier can be used for the purpose of the present invention.This carrier comprises:
-unique cloning site EcoRI
-metallothionein promoter,
-from west nile virus strain IS-98-ST1 geneome RNA 5' end noncoding region,
-translation initiation codon (ATG),
-from west nile virus strain IS-98-ST1 envelope E protein signal peptide (SEQ ID NO:15),
-from the geneome RNA 3' end noncoding region of west nile virus strain IS-98-ST1, two of which repetitive sequence and
3' end stem ring be deleted,
-S40polyA signal motif,
-unique cloning site ApaI.
1.2.SNAP clone
On template pMT/BiP/CHIK.sE2+SNAP label, use a pair 5'-SNAP as mentioned below by PCR
Carry out encoding the amplification of the DNA of SNAP protein sequence SEQ ID NO:2 with 3'-MCS primer.
Primer 5'-SNAP:5 '-aaaaaagatctgacaaagactgcgaaatg-3 ' (SEQ ID NO:7)
Primer 3'-MCS:5'-gaggagagggttagggataggcttacc-3'(SEQ ID NO:8)
Then digest PCR primer with Bgl II and Not I, and be inserted into DES system neutral plasmid p/MT/BiP/
Between unique Bgl II (at the 5' end of MCS) and Not I (at the 3' end of the MCS) site of V5-A.
The plasmid obtained is the pMT/BiP/SNAP-His label carrier of SEQ ID NO:9, including:
The insecticide ssBiP sequence of-SEQ ID NO:11,
The SNAP DNA sequence of-SEQ ID NO:1,
The enterokinase cleavage site point of-SEQ ID NO:12,
-EcoRV-Sma I restriction site,
-it is positioned at the coding His in restriction site downstream6The DNA (SEQ ID NO:14) of label and
-be positioned at i) between enhancer sequence and EcoRV-SmaI restriction site, and ii) EcoRV-SmaI restriction site
With coding His6Two DNA intervening sequences of the SEQ ID NO:13 between the DNA of label.
PMT/BiP/SNAP-His label carrier can available from pUC57 skeleton, and obtain there is sequence SEQ ID NO:10
Carrier, carrier includes:
-unique cloning site EcoRI
-metallothionein promoter
-from west nile virus strain IS-98-ST1 geneome RNA 5' end noncoding region,
-translation initiation codon,
The signal peptide of the envelope E protein from west nile virus strain IS-98-ST1 of-SEQ ID NO:15,
The SNAP DNA sequence of-SEQ ID NO:47,
-for exogenous array inserted the unique cloning site EcoR V in framework and Sma I/Xma I,
The enterokinase cleavage site point of-SEQ ID NO:12, its between SNAP enhancer DNA and cloning site,
-coding six His-Tag sequence DNA (SEQ ID NO:14),
-be positioned at i) between enhancer sequence and EcoRV-SmaI restriction site, and ii) EcoRV-SmaI restriction site
With coding His6Two DNA intervening sequences of the SEQ ID NO:13 between the DNA of label
-two translation termination codons,
-from the 3' end noncoding region of west nile virus strain IS-98-ST1 geneome RNA, two of which repetitive sequence and
3' end stem ring be deleted,
-S40polyA signal motif and
-unique cloning site ApaI.
PMT/BiP sample/SNAP-His label carrier can be available from pUC57 skeleton, and wherein SEQ ID NO:59 (is also shown in Fig. 8 A
And such as 8B) be inserted between unique site EcoR V and Hind III.Carrier has sequence SEQ ID NO:64.Comprising:
-unique cloning site EcoRI
-metallothionein promoter
-from west nile virus strain IS-98-ST1 geneome RNA 5' end noncoding region,
-translation initiation codon,
The signal peptide of the envelope E protein from west nile virus strain IS-98-ST1 of-SEQ ID NO:15,
The SNAP DNA sequence of-SEQ ID NO:47,
-for exogenous array inserted unique cloning site Bam H1, EcoR V, Apa I and Xma I in framework,
Two proTEV cleavage sites of-SEQ ID NO:52, between SNAP enhancer DNA and His label,
-coding six His-Tag sequence DNA (SEQ ID NO:14),
-be positioned at i) between enhancer sequence and EcoRV-SmaI restriction site, and ii) Apa I restriction site and volume
Code His6Two DNA intervening sequences of the SEQ ID NO:13 between the DNA of label
-two translation termination codons,
-from the 3' end noncoding region of west nile virus strain IS-98-ST1 geneome RNA, two of which repetitive sequence and
3' end stem ring be deleted,
-S40polyA signal motif and
-unique cloning site ApaI.
1.3. the clone of interest genes
1.3.1. the nucleoprotein N of Rift Valley fever virus (RVF-N)
5'-N and the 3'-N' primer listed below the determining of cDNA to carrying out encoding RFV-N protein sequence is used by PCR
To mutation.
Primer 5'-N:
5'-aaaaaggcgcgccagggggtggcggatctgacaactatcaagagcttcgagtccagtttgctgctc
-3'(SEQ ID NO:17)
Primer 3'-N:
5'-aaaaaaccggtcaatgatgatgatgatgatgacttccaccgccggctgctgtcttgtaagcctgag
Cgg-3'(SEQ ID NO:18)
The most non-viral albumen, such as interferon IFN α 1 or granzyme M
It is used as people's IFN α 1 protein sequence of SEQ ID NO:32.
It is used as the human granular enzyme M protein sequence of SEQ ID NO:54.Granzyme M is chymotrypsin-like serine albumen
Enzyme, it preferentially cuts its substrate after Met or Leu.It is constitutive expression in the intrinsic effect natural killer cell activated.
This protease also has antiviral and antitumor properties (van Domselaar R. et al., The Journal of
Immunology 2010；Hu D. et al., The Journal of Biological Chemistry 2010).
1.4. the gene of encoding proteins is inserted into pMT/BiP/SNAP-His label or pMT/BiP sample/SNAP-His mark
Sign carrier, thus obtain pMT/BiP/SNAP-albumen-His label or pMT/BiP sample/SNAP-albumen-His label carrier
1.4.1.RVF.N
The PCR primer obtained in 1.3.1 is digested by BssHII and AgeI, and is inserted in 1.2 the linearisation obtained
Unique BssHII (at the 3' end of SNAP gene) of plasmid p/MT/BiP/SNAP-His label and AgeI is (shuttle plasmid MCS's
3' end) between site.
The plasmid obtained is such as p/MT/BiP/SNAP-RVF.N/His label (SEQ ID NO:19).
1.4.2. the ED III albumen of different banzi virus
In order to strengthen ELISA based on recombinant flavivirus antigen or the specificity of western blot test, E protein (EDIII)
Antigenic domains III seemingly plant promising method (Ludolfs et al., 2007).With the method for the present invention test from
Xi Niluo (WN), Usu figure (USU), Japanese encephalitis (JE), Ticks propagate encephalitis (TBE), dengue serotypes 1 to 4 (DEN-1 ,-2 ,-
3 ,-4) and the production of restructuring EDIII of Huang heat (YF) virus.
Zoonotic WN, USU and JE virus belongs to JE serum complexes (serocomplex).Fruit bat S2 induction type
Expression system (DES, Invitrogen) has been selected in spinal zooblast large-scale production from banzi virus
Individual EDIII.Encode the E protein total length Domain III from banzi virus WN, USU, JE, TBE, DEN-1 to DEN-4 and YF
Synthetic gene is listed in SEQ ID NO:23 to SEQ ID NO:31.In the 1.2 plasmid pMT/BiP/SNAP-His labels obtained,
ED III sequence is fused to the C end of SNAP albumen in the frame and produces fusion protein S NAP-EDIII.
1.4.3.IFNα
Obtain plasmid pMT/BiP/SNAP-IFN-His label (see Fig. 4 A to Fig. 4 F) and pMT/BiP sample/SNAP-IFN-
His label (refers to Fig. 8 A and Fig. 8 B).
1.4.4. granzyme M
Obtain plasmid pMT/BiP/SNAP-GrM-His label (referring to Fig. 6 A to 6C).
2. it is transfected in host cell
2.1. it is transfected in S2 cell
Allow SNAP-label protein as with HisLabelThe secretion fusion protein of ending carries out the gained plasmid produced
PMT/BiP/SNAP-albumen-HisLabelAmmonia benzyl penicillium sp is shown with selection marker thing pCO-Blast cotransfection to S2 cell generates
The stable S2/sSNAP-albumen-His of element resistanceLabelCell line.
In blender jar (spinner) (1000ml), stable S2 cell line heavy metal cadmium (Cd2+) of growth stimulates 10 days,
Concentration also purification is from the albumen of extracellular medium.
After hatching 10 days with heavy metal cadmium, observe in the cell conditioned medium of stable S2/sSNAP-albumen-His label point
The accumulation of the SNAP-label protein secreted.
With the sheep blood serum of anti-His label, immunoblotting assay make it possible to detect extracellular SNAP-label protein (see
Fig. 2 B, 3B, 4B, 6C).
2.2. it is transfected in HeLa cell
Plasmid pMT/BiP sample/SNAP-IFN-HisLabelIt is transfected in HeLa cell.
Using anti-SNAP antibody, immunoblotting assay makes it possible to detect extracellular SNAP label interferon (see Fig. 7 B).
3. the purification of recombiant protein
With the outer His label of metal chelation resin and HLPC method purifying cells and SNAP label protein.
And TOS-N 3.1.RVF-N
RVF-N
Associating Plate-Forme 5 Production de Prot é ines recombinants et d ' Anticorps
(Institute Pasteur), from Cd2+2 liters of S2/sSNAP-RVFV.N-His after stimulating 10 daysLabelCell conditioned medium obtains up to
High-purity SNAP-label RVF.N albumen of 97mg.
TOS-N
Associating Plate-Forme 5 Production de Prot é ines recombinants et d ' Anticorps
(Institute Pasteur), from Cd2+2 liters of S2/sSNAP-TOS.N-His after stimulating 10 daysLabelCell conditioned medium obtains up to
High-purity SNAP-label RVF.N albumen of 41mg.
The summary of table 1. level of production:
The most solvable INF α 1
By cutting (Novagen test kit) with enterokinase, discharge solvable INF α 1 albumen from SNAP label.
3.3. from the antigen of different banzi virus
Table 2. uses SNAP-label protein liquid storage (stock) that Drosophila expression system is secreted
EDIII: from domain antigenicity III (Dengue [DEN], Xi Niluo [WN], Japan's brain of banzi virus E protein
Scorching [JE], Usu figure [USU], Ticks propagate encephalitis [TBE], yellow heat [YF], Mu Lei encephalitis [MVE], Wei Saiersi Blang [WSL],
Sieve Theo, hereby card)
EctoM: from the ectodomain of the M albumen of I type dengue virus
N gene from RVF: the nucleoprotein N (main virus antigen) of Rift Valley fever virus
N gene from TOS: the nucleoprotein N (main virus antigen) of toscana virus
SE from WN: from the soluble form of the envelope E protein of west nile virus
SE2 from CHIK: from the soluble form of the Envelope 2 protein of Chikungunya virus
SNAP-IFNAI: and the Alpha-IFN 1 that SNAP merges.
3.4. the production of granzyme M
In 7 days, every L culture supernatant reclaims the SNAP-GrM albumen of 10mg.
After purification step, the SNAP-GrM (seeing Fig. 6 C) of three kinds of forms being detected, the GrM enzyme that correspond to coupling is carried out
The cutting of SNAP albumen.
Obviously, it means that the human protease after being produced by the inventive method is activated (seeing below).
The comparison of 4.SNAP-label protein
Use the immunoblotting assay detection extracellular of specific antibody (identifying protein of interest and/or His label labelling)
The substantive production of SNAP-label protein:
Immunoblotting assay uses the anti-His of sheep blood serumLabelDetection extracellular SNAP-label RVF.N albumen (Fig. 2 B).Anti-
The people of RVF.N and mouse immune serum specific recognition restructuring SNAP label RVF.N albumen.
The immunoblotting assay using specific mouse polyclonal serum shows, although sequence similarity level is high, and restructuring
Cross reactivity is not had between WN and USU EDIII.Therefore, the SNAP-coming from WNV, JE, USU of the solubility of secretion
EDIII is suitable as recombinant virus antigens, for the member of specific diagnosis JE serum complexes, because in lesser degree,
USU and JE virus is accredited as potential emerging arbovirus recently in Europe.
The activity of 5.SNAP-label recombiant protein
The solvable restructuring SNAP-IFN α I secreted by S2/SNAP-IFN α I cell of induction shows powerful resisting
The antiviral effect of CHIKV.
Induce latter 10 days, collect Cd2+S2/SNAP-IFN α I (#5 × 10^6 cell/ml) supernatant (5ml) stimulated.Pass through
The immunoblotting using anti-His tag antibody observes that in cell conditioned medium the accumulation of solvable SNAP-IFN α I protein (sees below
Literary composition).Have on the HeLa cell of Chikungunya virus of expressing luciferase (Luc) gene in infection, assessment SNAP-IFN α I's
Antiviral activity.Infect latter 6 hours and determine Luc activity.(Infergen (Infergen) is used as interior part to IFN alphacon 1
Analysis, it is known that the antiviral effect of its anti-CHIKV powerful in HeLa cell.Cd2+The S2/SNAP-Tos.N stimulated is (from torr
SIKA receives the N protein of virus) supernatant is as negative control.Figure shown in Fig. 4 C shows the SNAP-IFN α I or 0.1 μ g of 1 μ l secretion
Infergen can suppress CHIKV duplication in host cells infected.The dosage dependent effect of SNAP-IFN α is as shown in FIG. 4 C.
The Luc activity of 20% is still observed with 0.1 μ l solvable SNAP-IFN α I or 0.01 μ g Infergen.Under higher proof load,
SNAP-TOS-N is used not observe antiviral effect.
Once producing in S2 cell conditioned medium, granzyme M is activated
As it was previously stated, detect in transfection has the S2 cell conditioned medium of carrier pMT/BiP/SNAP-GrM-His label
Three kinds of forms (see Fig. 6 C) of SNAP-GrM.
These three form correspond to the cutting of the SNAP albumen that the GrM enzyme of coupling is carried out.SNAP really comprise three potential
GrM cleavage site (see Fig. 6 B).Showing with the immunoassay of the antibody of anti-His-or anti-SNAP, these three form is strictly
The fragment of the fusion protein S NAP-GrM of secretion.
Less form (35kDa) correspond to the GrM that in purge process, SNAP major part is deleted.
These results clearly illustrate, although coupling has SNAP albumen, GrM protease produced by present system be
Activated.
This is the most interesting, because it is known that activated human protease is difficult to by recombinant production.
Once produce in HEK 293 cell conditioned medium, hSULF-2ΔTMDIt is activated
hSULF-2ΔTMDExpress and purification has recombiant plasmid pcDNA3/De SNAPuniv-hSULF-2 in transfectionΔTMD's
HEK-293 cell (see Figure 13 A).
The hSULF-2 available from supernatant is evaluated according to followingΔTMDThe enzymatic activity of polypeptide:
HEK 293 cell transient transfection has pcDNA3/DeSNAP-hSULF2 Δ TMD.Two days later, the equal portions of cell conditioned medium
Hatch in 50mM Tris pH7.5 with 20mM non-fluorescence puppet substrate 4-methyl umbelliferone (Umbelliferone) (4-MUS),
20mM magnesium chloride (1:1, V/V) and enzyme (in conditioned medium) hatch 2-4 hour in 96-orifice plate at 37 DEG C.By adding
Add (1:1v/v) 1.5M NaHCO3/Na2CO3pH 10.5 and terminate enzyme reaction, and monitor 4-by exometer (exciting: 360nm)
The generation of methyl umbelliferone fluorescence-causing substance.The value of SULF activity in cell conditioned medium is measured with the optical density (OD) at 460nm.
It is interesting that as shown in Figure 13 B, the albumen (coupling has SNAP) of secretion is activated.
The stability of 6.SNAP label recombiant protein
It has been surprisingly observed that, including the fusion protein of SNAP peptide, ratio is not when having SNAP peptide, the most more stable.
Highly purified CHIK.sE2-SNAP, SNAP-WN.EDIII and SNAP-IFNAI albumen, in aseptic PBS
During 0.1mg/ml (Vol:0.1ml) unsaturation concentration, hatch at-80 DEG C, 4 DEG C, 25 DEG C or 37 DEG C 4 days, or mutually synthermal
Under hatch two months.
SDS-PAGE 4-12% separates albumen sample (1 μ g), and uses PageBlue protein staining solution
(Fermentas) visualize with Coomassie brilliant G-250 dyestuff.
Figure 10 A to 10D discloses the result that the vitro stability by comparing three kinds of different fusion protein obtains.
It is essential that at 4 DEG C after 2 months, and after at room temperature hatching four days at (25 DEG C) or 37 DEG C, all fusions
Albumen is the most complete.
Particularly, under body temperature (37 DEG C), after 4 days, IFN is unaffected, and still observes after 2 months at 37 DEG C
Arrive, therefore have SNAP, internal stability still may highly improve by its coupling.
The vitro detection of the SNAP-RVF.N that 7.S2 cell produces
It is used as diagnostic tool, for detecting the Sheep Blood of infection according to the SNAP-RVF.N fusion protein that above-mentioned code produces
Anti-RVF.N antibody in Qing.
Detect these fusion protein, and compare (the many things of RVF from BDSL with from IdVet with commercial detection test kit
The RVF IgG detection kit planted).
Testing on 46 parts of sheep serums, sheep immunity inoculation has RVF vaccine.SNAP-RVF.N fusion protein is direct
It is coated at the bottom of hole, or through biotinylation and join in the coated hole of Streptavidin.
By indirect ELISA method, direct coated is used to have high-purity recombinant antigen SNAP-RVF.N of 0.2 μ g in PBS (dense
Degree: microtitration 96-hole speckle 2 μ g albumen/ml), overnight detects anti-RVF antibody at 4 DEG C.After saturated, the serum of dilution
Hatch with SNAP-RVF.N.Coupling has the peroxidase of sheep anti-igg to resist as two.Carry out with Peroxidase Substrate System
ELISA, measures optical density (OD) at 450nm.If OD is the twice of the OD from NIS, then it is assumed that sample serum
It is positive.
It is interesting that result shows when in direct coated to hole, compared to business albumen, SNAP-RVF.N fusion protein
Provide identical sensitivity and specificity (not shown).When albumen is biotinylated, the repeatability of result is relatively low.
Available from autarcetic Ox blood serum, achieve identical result (data are not shown).
These results show that the fusion protein of the present invention can be used as identifying that in biological specimen, virus infects or antibacterial sense
The diagnostic tool of dye.
The most multiple immunoassay based on pearl
In the context of the present invention, multiple immunoassay based on pearl is developed for quick and synchronous detecting biological fluid
In anti-arboviral antibody.
This system is based on xMAP technology (Luminex company), and use is coated with the mixture of microspheres of antigen as specific
The trapping agent of human normal immunoglobulin.With the microsphere of the MGMT fusion protein coupling difference group of purification, (Magplex, Luminex are public
Department), i.e. 3.3 joint described in SNAP-label virus recombiant protein: sSNAP-DV1.EDIII, sSNAP-DV2.EDIII,
sSNAP-DV3.EDIII、sSNAP-DV4.EDIII、sSNAP-WN.EDIII、sSNAP-JE.EDIII、sSNAP-
USU.EDIII、sSNAP-TBE.EDIII、sSNAP-YF.EDIII、sSNAP-MVE.EDIII、sSNAP-Rocio.EDIII、
SSNAP-WSL.EDIII, sSNAP-ZIKA.EDIII, SNAP-DV1ectoM, sSNAP-N.RVF, sSNAP-N.TOS and
CHIK.sE2-SNAP.With the substrate of mgmt protein as joint (BG-PEG-NH2, New England Biolabs), will restructuring
Antigen is covalently coupled to carboxyl microsphere surface, thus compared with standard amine coupling procedure, strengthens antibody capture efficiency.
The technical identification using anti-SNAP-tag antibody and specific mouse monoclonal antibody confirms coupling efficiency, and
Indicate long-term Antigen Stability (up to 6 months).The application is not limited to virus antigen, because any peptide or polypeptide can be used for
Being coated and antibody capture subsequently of pearl.
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Harlowe, et al., 1988, Antibodies:A Laboratory Manual, Cold Spring Harbor
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(referred to herein as " Sambrook et al., 1989 ")；
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Claims (44)
1. an expression vector, it is for expressing recombiant protein in host cell, and described expression vector is included in single opening
Reading frame encodes following nucleotide sequence, according to the direction of 5' to 3':
A) there is in described host cell the peptide secretion signal of function,
B) 6-methyl guanine-DNA-transmethylase (MGMT, EC 2.1.1.63), its catalyst structure domain or mutant, and
C) recombiant protein.
Expression vector the most according to claim 1, wherein said MGMT enzyme is albumen or its homology of SEQ ID NO:4
Thing.
Expression vector the most according to claim 1, wherein said MGMT mutant be SEQ ID NO:2 SNAP albumen or
Its congener.
4. according to the expression vector described in any one of claims 1 to 3, wherein said open reading frame operationally with induction type
Promoter combines, and described inducible promoter has function in identical host cell as peptide secretion signal.
5., according to the expression vector described in any one of Claims 1-4, wherein said peptide secretion signal and described induction type start
Son, in spinal zooblast, preferably in insect cell, more preferably has function in Drosophila S 2 cells.
6., according to the expression vector described in any one of claim 1 to 5, wherein said peptide secretion signal and described induction type start
Son is in vertebrate cells, preferably in mammalian cell, more preferably at Chinese hamster ovary celI, YB2/O cell, COS cell, HEK
Cell, NIH3T3 cell, HeLa cell or derivatives thereof there is function.
7., according to the expression vector described in any one of claim 1 to 6, wherein said recombiant protein is selected from diagnosis and/or treatment
Albumen and/or polypeptide.
Expression vector the most according to claim 7, wherein said diagnosis and treatment albumen and/or polypeptide are selected from:
-antibacterial or viral immunogenic albumen, more preferably propagate encephalitis, yellow heat, Usu figure, sieve from Dengue, Japanese encephalitis, Ticks
The EDIII albumen of Theo, Mu Lei encephalitis, Wei Saiersi Blang, Zi Ka or west nile virus, or from Rift Valley fever or Tuscany
The nucleoprotein N of virus, or the soluble form of the E2 envelope protein from Chikungunya virus, or west nile virus E envelope protein
Soluble form, and
-blood factor, anticoagulant, somatomedin, hormone, therapeutic enzyme and monoclonal antibody and cytokine,
-anti-tumor protein,
-microorganism, virus and/or parasite polypeptide,
-antigen, such as cancer antigen.
Expression vector the most according to claim 7, wherein said diagnosis and treatment albumen selected from IFN-α, granzyme M,
FasL、SSX2、NERCMSL、hSULF2ΔTMDAnd CNTN4.
10., according to the expression vector described in any one of claim 1 to 9, wherein said MGMT enzyme is by SEQ ID NO:3 or SEQ
The DNA sequence encoding of ID NO:68.
11. according to the expression vector described in any one of claim 1 to 9, and wherein said MGMT enzyme mutant is by SEQ ID NO:1
Or the DNA sequence encoding of SEQ ID NO:47.
12. according to the expression vector described in any one of claim 1 to 11, and it also encodes at least one peptide cleavage site, and it is excellent
Bit selecting is in MGMT enzyme, between its mutant or catalyst structure domain and recombiant protein.
13. according to the expression vector described in any one of claim 1 to 12, and it also encodes a labelling, and it is preferably placed at restructuring
The C end of albumen.
14. according to the expression vector described in any one of claim 1 to 13, and it goes back encoded interval sequence, is preferably placed at described
Between MGMT enzyme, its mutant or catalyst structure domain and described recombiant protein, and/or described recombiant protein and described labelling it
Between.
15. expression vectors according to claim 1, its coding:
-peptide BiP insect signal,
The SNAP albumen of-SEQ ID NO:2,
Recombiant protein defined in-claim 9,
-enterokinase peptide cleavage site,
-polyhistidine labelling, and
Article-two, there is the intervening sequence of aminoacid sequence Gly-Gly-Gly-serine.
16. expression vectors according to claim 1, its coding:
-BiP sample peptide signal,
The SNAP albumen of-SEQ ID NO:2,
Recombiant protein defined in-claim 9,
-proTEV peptide cleavage site,
-polyhistidine labelling, and
Article-two, there is the intervening sequence of aminoacid sequence Gly-Gly-Gly-serine.
17. 1 kinds of expression vectors being used for expressing recombiant protein in host cell, it is included in single open reading frame volume
The nucleotide sequence that code is following, according to the direction of 5' to 3':
A) BiP sample peptide signal,
B) mgmt protein of SEQ ID NO:4 or the SNAP albumen of SEQ ID NO:2,
C) two proTEV peptide cleavage sites,
D) polyhistidine labelling, and
E) two intervening sequences with aminoacid sequence Gly-Gly-Gly-serine.
18. 1 kinds of reconstitution cells, the expression that its stable transfection has plasmid, described plasmid to comprise any one of claim 1 to 17 carries
Body, described plasmid preferably comprises SEQ ID NO:59 or SEQ ID NO:69.
19. reconstitution cells according to claim 18, wherein said reconstitution cell is antibacterial, preferably Bacillus coli cells.
20. reconstitution cells according to claim 18, wherein said reconstitution cell is antibacterial, insect cell, more preferably fruit
Fly S2 cell or derivatives thereof；Mammalian cell, more preferably Chinese hamster ovary celI, YB2/O cell, COS cell, HEK cell,
NIH3T3 cell, HeLa cell, PER.C6 cell or derivatives thereof；With avian cell such as EBx cell, preferably EB66 cell or its
Derivant.
Reconstitution cell described in 21. any one of claim 18 to 20 is used for producing defined in any one of claim 1 to 17
The purposes of expression vector.
Reconstitution cell described in 22. any one of claim 18 to 20 is for producing the purposes of recombiant protein.
23. 1 kinds of fused polypeptide, comprising:
Peptide secretion signal and
6-methyl guanine-DNA-transmethylase, its mutant or catalyst structure domain.
24. fused polypeptide according to claim 23, wherein said MGMT mutant is the SNAP albumen of SEQ ID NO:2
Or its congener.
25. fused polypeptide according to claim 23, wherein said MGMT enzyme is albumen or its homology of SEQ ID NO:4
Thing.
26. according to the fused polypeptide described in any one of claim 23 to 25, and it is additionally included in MGMT enzyme, its mutant or catalysis
The recombiant protein of the C end of domain.
27. fused polypeptide according to claim 26, also include a labelling, preferably polyhistidine labelling, and it is positioned at
Recombinant protein c end.
28. 1 kinds of spinal animal reconstitution cells, its stable transfection is had the right the table described in any one of requirement 1 to 5 and 7 to 14
Reach carrier.
29. spinal animal reconstitution cells according to claim 28, wherein it is insect cell.
30. spinal animal reconstitution cells according to claim 29, wherein it is Drosophila melanogaster Drosophila
Melanogaster cell, more preferably Drosophila S 2 cells.
31. spinal animal reconstitution cells according to claim 30, it is characterised in that:
I) described expression vector is selected from:
-include SEQ ID NO:19 carrier or the nucleotide sequence that is cloned in cell, cell on August 19th, 2010 with
Numbering CNCM I-4357 be deposited in France country Organism Depositary, Institute Pasteur, Paris, France,
-include SEQ ID NO:22 the carrier of claim 7 or the nucleotide sequence that is cloned in cell, cell is in 2010
On October 27, is deposited in the national Organism Depositary of France with numbering CNCM I-4381, Institute Pasteur,
-include SEQ ID NO:21 the carrier of claim 7 or the nucleotide sequence that is cloned in cell, cell is in 2010
On October 27, is deposited in the national Organism Depositary of France with numbering CNCM I-4382, Institute Pasteur,
-include SEQ ID NO:9 carrier or the nucleotide sequence that is cloned in cell, cell in JIUYUE in 2010 29 days with
Numbering CNCM I-4368 be deposited in France country Organism Depositary, Institute Pasteur, and
-include SEQ ID NO:20 the carrier of claim 7 or the nucleotide sequence that is cloned in cell, cell is in 2010
On JIUYUE 29, is deposited in the national Organism Depositary of France with numbering CNCM I-4369, Institute Pasteur,
-include the carrier of claim 17 of SEQ ID NO:10 or 59 or 69,
The carrier of-SEQ ID NO:64 or the nucleotide sequence being cloned in cell, cell in December in 2011 9 days with numbering
CNCM I-4581 be deposited in France country Organism Depositary, Institute Pasteur,
The carrier of-SEQ ID NO:71,
-include SEQ ID NO:55, SEQ ID NO:57 or 72 or 74, SEQ ID NO:77,79 or 81, SEQ ID NO:89,
The carrier of the claim 9 of SEQ ID NO:84 or 86, SEQ ID NO:92 or SEQ ID NO:96,
Or
Ii) it is selected from:
-it being deposited in country of France Organism Depositary on August 19th, 2010 with numbering CNCM I-4357, Pasteur is studied
Cell,
-it being deposited in country of France Organism Depositary on October 27th, 2010 with numbering CNCM I-4381, Pasteur is studied
Cell,
-it being deposited in country of France Organism Depositary on October 27th, 2010 with numbering CNCM I-4382, Pasteur is studied
Cell,
-within 29th, it being deposited in country of France Organism Depositary with numbering CNCM I-4368 in JIUYUE in 2010, Pasteur is studied
Cell,
-within 29th, it being deposited in country of France Organism Depositary with numbering CNCM I-4369 in JIUYUE in 2010, Pasteur is studied
Cell,
-within 5th, it being deposited in country of France Organism Depositary with numbering CNCM I-4565 in December in 2011, Pasteur is studied
Cell,
-within 5th, it being deposited in country of France Organism Depositary with numbering CNCM I-4566 in December in 2011, Pasteur is studied
Cell,
-within 5th, it being deposited in country of France Organism Depositary with numbering CNCM I-4567 in December in 2011, Pasteur is studied
Cell,
-within 5th, it being deposited in country of France Organism Depositary with numbering CNCM I-4568 in December in 2011, Pasteur is studied
Cell,
-within 5th, it being deposited in country of France Organism Depositary with numbering CNCM I-4569 in December in 2011, Pasteur is studied
Cell,
-within 5th, it being deposited in country of France Organism Depositary with numbering CNCM I-4570 in December in 2011, Pasteur is studied
Cell,
-within 5th, it being deposited in country of France Organism Depositary with numbering CNCM I-4571 in December in 2011, Pasteur is studied
Cell,
-within 5th, it being deposited in country of France Organism Depositary with numbering CNCM I-4572 in December in 2011, Pasteur is studied
Cell,
-within 8th, it being deposited in country of France Organism Depositary with numbering CNCM I-4576 in December in 2011, Pasteur is studied
Cell,
-within 8th, it being deposited in country of France Organism Depositary with numbering CNCM I-4577 in December in 2011, Pasteur is studied
Cell,
-within 8th, it being deposited in country of France Organism Depositary with numbering CNCM I-4578 in December in 2011, Pasteur is studied
Cell,
-within 8th, it being deposited in country of France Organism Depositary with numbering CNCM I-4579 in December in 2011, Pasteur is studied
Cell,
-within 8th, it being deposited in country of France Organism Depositary with numbering CNCM I-4580 in December in 2011, Pasteur is studied
Cell,
-within 9th, it being deposited in country of France Organism Depositary with numbering CNCM I-4583 in December in 2011, Pasteur is studied
Cell,
-within 9th, it being deposited in country of France Organism Depositary with numbering CNCM I-4584 in December in 2011, Pasteur is studied
Cell,
-within 9th, it being deposited in country of France Organism Depositary with numbering CNCM I-4585 in December in 2011, Pasteur is studied
Cell, and
-within 9th, it being deposited in country of France Organism Depositary with numbering CNCM I-4586 in December in 2011, Pasteur is studied
Cell.
32. 1 kinds of vertebrates reconstitution cells, its stable transfection is had the right the table of any one of requirement 1 to 4,6 to 14 and 16 to 17
Reach carrier.
33. vertebrates reconstitution cells according to claim 32, wherein it is mammalian cell, preferably Chinese hamster ovary celI,
YB2/O cell, COS cell, HEK cell, NIH3T3 cell, HeLa cell or derivatives thereof.
34. according to the vertebrates reconstitution cell described in claim 32 or 33, it is characterised in that:
When protein of interest is IFN α, described expression vector is the load of the claim 9 including SEQ ID NO:57 or 72 or 74
Body；
When protein of interest is cancer antigen SSX2, described expression vector is to include SEQ ID NO:77,79 or the claim 9 of 81
Carrier；
When protein of interest is granzyme M, described expression vector is the carrier of the claim 9 including SEQ ID NO:55；
When protein of interest is FasL, described expression vector is the carrier of the claim 9 including SEQ ID NO:89；
When protein of interest is cancer antigen NERCMSL, described expression vector is to include SEQ ID NO:84 or the claim 9 of 86
Carrier；Or
When protein of interest is contactin CNTN4, described expression vector includes SEQ ID NO:92；Or
When protein of interest is hSULF2ΔTMDTime, described expression vector is the carrier of the claim 9 including SEQ ID NO:96.
35. 1 kinds of methods strengthening expression of recombinant proteins, it includes by described albumen and peptide secretion signal, together with enzyme 6-methyl bird
Purine-DNA-transmethylase, its mutant or catalyst structure domain coexpression, described coexpression is preferably at spinal zooblast
In carry out.
36. methods according to claim 35, wherein said MGMT enzyme is the albumen of SEQ ID NO:4.
37. methods according to claim 35, wherein said MGMT mutant enzyme be SEQ ID NO:2 SNAP albumen or
Its congener.
38. 1 kinds produce the methods of recombiant protein in cell is cultivated, and it includes step:
A) expression vector described in any one of claim 1 to 16 is provided,
B) described expression vector is introduced host cell,
C) nucleotide making the described host cell of introducing carries out expression to produce described recombiant protein.
39. according to the method described in any one of claim 35 to 38, and wherein the expression of recombiant protein is the cell reclaimed
At least 40mg/L in culture supernatant or more than.
40.6-methyl guanine-DNA-transmethylase, its mutant or catalyst structure domain are used for strengthening heterologous protein to be infected
There is the purposes produced in the host cell of replication form or defective vector.
41. purposes according to claim 40, wherein said MGMT enzyme is the albumen of SEQ ID NO:4.
42. purposes according to claim 40, wherein said MGMT mutant is the SNAP albumen of SEQ ID NO:2.
43.6-methyl guanine-DNA-transmethylase, its mutant or catalyst structure domain, as being fused to or be connected to weight
The protection polypeptide of histone, for improving recombiant protein in storage medium, half-life in blood plasma or buffer, is used for improving
As medicine or the half-life of the recombiant protein of vaccine, or for improving the half-life of the recombiant protein being used in diagnostic kit
Purposes.
44. purposes according to claim 43, wherein said recombiant protein is selected from: insulin, IFN α, granzyme M,
SSX2, FasL, hSULF, contactin and NERMCSL.
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| US9109219B2 (en) | 2015-08-18 |
| EP2649178A1 (en) | 2013-10-16 |
| JP6316856B2 (en) | 2018-04-25 |
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| EP3202897A1 (en) | 2017-08-09 |
| US10017769B2 (en) | 2018-07-10 |
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| CA2819552C (en) | 2023-09-26 |
| BR112013014457A8 (en) | 2017-12-19 |
| JP2016154543A (en) | 2016-09-01 |
| US20160017367A1 (en) | 2016-01-21 |
| ES2627117T3 (en) | 2017-07-26 |
| US20130309747A1 (en) | 2013-11-21 |
| EP3202897B1 (en) | 2020-04-15 |
| CN103476928B (en) | 2017-03-29 |
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